Allergy vaccines

ABSTRACT

The invention provides methods and materials related to vaccines against self polypeptides. For example, the invention provides compositions containing chimeric IgE polypeptides and adjuvants.

RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. ProvisionalApplication Serial No. 60/408,648, filed Sep. 5, 2002.

BACKGROUND

[0002] 1. Technical Field

[0003] The invention relates to methods and materials involved in theuse of vaccines containing a polypeptide (e.g., a chimeric IgEpolypeptide) and an adjuvant. Such vaccines can be used to elicit ananti-self response (e.g., an anti-self IgE response).

[0004] 2. Background Information

[0005] During the past few decades several diseases caused bymalfunctions of the immune system have become the major challenges ofmodem day medicine. Two such areas are the allergic and autoimmunediseases. Allergies have become almost epidemic during the past 20-30years. Estimations range from 20-30 percent of the total populationbeing affected. Atopic allergies, or IgE mediated allergies, are thedominating form.

[0006] Common types of atopic allergies include hay fever, furallergies, dust mite allergies, insect venom allergies, extrinsicasthma, and many types of food allergies. An interesting question iswhether vaccines can be developed against these types of diseases.Hyposensitization therapy has been used to treat allergies since thebeginning of the twentieth century (Noon, Lancet, 1:1572 (1911); andFreeman, Lancet, 1:1178 (1914)). This is an allergen-dependent treatmentstrategy, which involves the use of allergen extracts to treat patientsby injection. Hyposensitization therapy has, however, been questioneddue to often low efficacy and sometimes severe side effects. Inaddition, different extracts must be used for each individual form ofallergy. New strategies to treat allergies thus are presently beingevaluated.

[0007] Vaccines are typically administered with an adjuvant such asalum. Alum, however, is a relatively weak potentiator of cell-mediatedimmune responses (Krishnan et al., Infect. Immun., 68:54-63 (2000) andGupta et al., Adjuvant properties of aluminum and calcium compounds, p.229-248. In M. F. Powell and M. J. Newman (ed.), Vaccine design: thesubunit and adjuvant approach. Plenum Press, New York, N.Y. (1995)). Inaddition, aluminum hydroxide has been reported to attract eosinophils tothe site of injection and increase the levels of antigen-specific andtotal IgE antibodies that may promote IgE-mediated allergic reactions(Baylor et al., Vaccine, 20:S18-S23 (2002); Walls, Proc. Soc. Exp. Biol.Med., 156:431-435 (1977); and Nagel et al., J. Immunol., 118:334-341(1977)).

SUMMARY

[0008] The invention provides materials and methods related to vaccinesagainst self polypeptides. For example, the invention providescompositions containing a polypeptide (e.g., a chimeric IgE polypeptide)and an adjuvant. The polypeptide typically contains self and non-selfcomponents, which can result in both anti-self and anti non-self immuneresponses when administered to a mammal. For example, when administeredto a mammal, the chimeric IgE polypeptides provided herein can reducethe IgE antibody effects of IgE-related diseases such as asthma,allergies, and eczema. The adjuvant typically is selected to give arelatively high anti-self response, as compared to compositionscontaining other adjuvants.

[0009] The invention is based on the discovery that chimeric IgEpolypeptides in combination with an adjuvant can be used to reduce thelevel of detectable free IgE antibodies in a mammal. For example,administration of chimeric IgE polypeptides in combination with aluminumcompounds unexpectedly resulted in a reduction in the levels ofdetectable free IgE antibodies despite previous reports that aluminumcompounds increase total IgE levels.

[0010] In general, one aspect of the invention features a compositioncontaining a polypeptide (e.g., an ORO polypeptide or an OSOpolypeptide) and alum, wherein the polypeptide contains a self IgEpolypeptide sequence, and wherein administration of the composition to amammal produces an anti-self IgE antibody response with a titerdilution₅₀ value greater than 100. The composition can contain betweenabout ten micrograms and about one gram of the polypeptide. Thecomposition can contain about 280 micrograms of the polypeptide. Thecomposition can contain between about ten microliters and about onemilliliter of alum. The composition can contain about 50 microliters ofalum. The titer dilutions₅₀ value can be greater than 150, greater than200, or greater than 400.

[0011] In another embodiment, the invention features a compositioncontaining a polypeptide (e.g., an ORO polypeptide or an OSOpolypeptide) and MN51, wherein the polypeptide contains a self IgEpolypeptide sequence, and wherein administration of the composition to amammal produces an anti-self IgE antibody response with a titerdilution₅₀ value greater than 100. The composition can contain betweenabout ten micrograms and about one gram of the polypeptide. Thecomposition can contain about 100 micrograms of the polypeptide. Thecomposition can contain between about ten microliters and about onemilliliter of MN51. The composition can contain about 50 microliters ofMN51. The titer dilutions₅₀ value can be greater than 150, greater than200, or greater than 400.

[0012] Another embodiment of the invention features a compositioncontaining alum and about 280 micrograms of a polypeptide (e.g., an OROpolypeptide or an OSO polypeptide).

[0013] In another embodiment, the invention features a compositioncontaining MN51 and at least about 100 micrograms of a polypeptide(e.g., an ORO polypeptide or an OSO polypeptide).

[0014] In another aspect, the invention features a method for inducingan anti-self IgE antibody response in a mammal, the method includingadministering to the mammal a composition under conditions wherein themammal produces an anti-self IgE antibody response with a titerdilutions₅₀ value greater than 100, wherein the composition contains apolypeptide and alum, and wherein the polypeptide contains a selfpolypeptide sequence from an IgE polypeptide.

[0015] In another embodiment, the invention features a method forinducing an anti-self IgE antibody response in a mammal, the methodcontaining administering to the mammal a composition under conditionswherein the mammal produces an anti-self IgE antibody response with atiter dilutions₅₀ value greater than 100, wherein the compositioncontains a polypeptide and MN51, and wherein the polypeptide contains aself IgE polypeptide sequence from an IgE polypeptide.

[0016] In another embodiment, the invention features methods forinducing a reversible anti self-IgE response in a mammal (e.g., aprimate such as a monkey or human). Such methods involve administering apolypeptide having a self IgE sequence to said mammal under conditionswherein the mammal mounts an antibody response to self-IgE in a mannersuch that the response peaks and then decreases with time. For example,the anti self-IgE response can be a primary response that decreases withtime (e.g., decreases to undetectable levels within 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, or more months).

[0017] Another embodiment of the invention features methods for inducingan anti self-IgE response in a mammal (e.g., a primate such as a monkeyor human) after said mammal has experienced a primary anti self-IgEresponse. Such methods involve administering a polypeptide having a selfIgE sequence to the mammal under conditions wherein the mammal mounts anantibody response to self-IgE in a manner consisted with a secondaryantibody response.

[0018] Another embodiment of the invention features methods for inducinga series of anti self-IgE responses in a mammal (e.g., a primate such asa monkey or human). Such methods involve administering a polypeptidehaving a self IgE sequence to the mammal at different times and underconditions wherein the mammal mounts a detectable anti self-IgE responsethat peaks within at least one year (e.g., within at least 11, 10, 9, 8,7, 6, 5, 4, 3, 2, or 1 month) of each administration.

[0019] Another embodiment of the invention features a compositioncontaining a polypeptide and an aluminum compound, wherein thepolypeptide contains a self IgE polypeptide sequence, and whereinadministration of the composition to a mammal reduces the level ofdetectable free IgE in the mammal. The polypeptide can be a chimeric IgEpolypeptide. The polypeptide can contain a sequence set forth in SEQ IDNO:3, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ IDNO:16, SEQ ID NO:18, or SEQ ID NO:21. The composition can containbetween about ten micrograms and about one gram of the polypeptide. Thecomposition can contain about 280 micrograms of the polypeptide. Thealuminum compound can be an aluminum hydrogel compound. The aluminumcompound can be alum. The composition can contain between about tenmicroliters and about one milliliter of the alum. The composition cancontain about 50 microliters of the alum. The reduction can be at leastabout a 10 percent reduction (e.g., at least about a 20, 30, 40, 50, 60,70, 80, 90, or 95 percent reduction). The reduction can be a reductionfrom about 10 percent to about 95 percent (e.g., from about 20 percentto about 95 percent, from about 25 percent to about 95 percent, fromabout 50 percent to about 95 percent, from about 75 percent to about 95percent, from about 85 percent to about 95 percent, from about 25percent to about 80 percent, or from about 50 percent to about 80percent). The reduction can be detectable in an ELISA. An IgE receptorpolypeptide sequence can be used in the ELISA. The administration of thecomposition to the mammal can produce an anti self IgE antibody responsewith a titer dilution₅₀ value greater than 100 (e.g., greater than 200,300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, or1500).

[0020] Another embodiment of the invention features a compositioncontaining a polypeptide and MN51, wherein the polypeptide contains aself IgE polypeptide sequence, and wherein administration of thecomposition to a mammal reduces the level of detectable free IgE in themammal. The polypeptide can be a chimeric IgE polypeptide. Thepolypeptide can contain a sequence set forth in SEQ ID NO:3, SEQ IDNO:6, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ IDNO:18, or SEQ ID NO:21. The composition can contain between about tenmicrograms and about one gram of the polypeptide. The composition cancontain about 100 micrograms of the polypeptide. The composition cancontain between about ten microliters and about one milliliter of theMN51. The composition can contain about 50 microliters of the MN51. Thereduction can be at least about a 10 percent reduction (e.g., at leastabout a 20, 30, 40, 50, 60, 70, 80, 90, or 95 percent reduction). Thereduction can be a reduction from about 10 percent to about 95 percent(e.g., from about 20 percent to about 95 percent, from about 25 percentto about 95 percent, from about 50 percent to about 95 percent, fromabout 75 percent to about 95 percent, from about 85 percent to about 95percent, from about 25 percent to about 80 percent, or from about 50percent to about 80 percent). The reduction can be detectable in anELISA. An IgE receptor polypeptide sequence can be used in the ELISA.The administration of the composition to the mammal can produce an antiself IgE antibody response with a titer dilution₅₀ value greater than100 (e.g., greater than 200, 300, 400, 500, 600, 700, 800, 900, 1000,1100, 1200, 1300, 1400, or 1500).

[0021] Another embodiment of the invention features a compositioncontaining an aluminum compound and about 30 to 300 micrograms of achimeric IgE polypeptide.

[0022] Another embodiment of the invention features a compositioncontaining MN51 and about 30 to 300 micrograms of a chimeric IgEpolypeptide.

[0023] Another embodiment of the invention features a method forinducing an anti self IgE antibody response in a mammal. The methodincludes administering to the mammal a composition under conditionswherein the mammal reduces the level of detectable free IgE in themammal, wherein the composition contains a polypeptide and an aluminumcompound, and wherein the polypeptide contains a self polypeptidesequence. The polypeptide can contain an amino acid sequence set forthin SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14,SEQ ID NO:16, SEQ ID NO:18, or SEQ ID NO:21.

[0024] Another embodiment of the invention features a method forinducing an anti self IgE antibody response in a mammal. The methodincludes administering to the mammal a composition under conditionswherein the mammal reduces the level of detectable free IgE in themammal, wherein the composition contains a polypeptide and MN51, andwherein the polypeptide contains a self polypeptide sequence. Thepolypeptide can contain an amino acid sequence set forth in SEQ ID NO:3,SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQID NO:18, or SEQ ID NO:21.

[0025] Another embodiment of the invention features a method forinducing a reversible anti self-IgE response in a primate. The methodincludes administering a polypeptide having a self IgE sequence to theprimate under conditions wherein the primate mounts an antibody responseto self-IgE that peaks and then decreases with time. The primate can bea monkey. The antibody response to self-IgE can be a primary responsethat decreases with time. The antibody response to self-IgE can decreaseto undetectable levels within nine months of the administration. Thepolypeptide can contain a sequence set forth in SEQ ID NO:6, SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, or SEQ IDNO:21.

[0026] Another embodiment of the invention features a method forinducing an anti self-IgE response in a mammal after the mammal hasexperienced a primary anti self-IgE response. The method includingadministering a polypeptide having a self IgE sequence to the mammalunder conditions wherein the mammal mounts an antibody response toself-IgE in a manner consistent with a secondary antibody response. Themammal can be a primate. The polypeptide can contain a sequence setforth in SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:12, SEQ IDNO:14, SEQ ID NO:16, SEQ ID NO:18, or SEQ ID NO:21.

[0027] Another embodiment of the invention features a method forinducing a series of anti self-IgE responses in a mammal. The methodincluding administering a polypeptide having a self IgE sequence to themammal at different times and under conditions wherein the mammal mountsa detectable anti self-IgE response that peaks within at least one yearof each administration. The mammal can mount a detectable anti self-IgEresponse that peaks within at least three months of each administration.

[0028] Unless otherwise defined, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention pertains. Although methods andmaterials similar or equivalent to those described herein can be used topractice the invention, suitable methods and materials are describedbelow. All publications, patent applications, patents, and otherreferences mentioned herein are incorporated by reference in theirentirety. In case of conflict, the present specification, includingdefinitions, will control. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting.

[0029] The details of one or more embodiments of the invention are setforth in the accompanying drawings and the description below. Otherfeatures, objects, and advantages of the invention will be apparent fromthe description and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

[0030]FIG. 1 is a diagram of the nucleic acid vector designatedpRES-ORO.

[0031]FIG. 2 is a nucleic acid sequence listing of the pRES-ORO vector(SEQ ID NO:1).

[0032]FIG. 3 is a nucleic acid sequence listing of an insert sequencethat encodes an ORO polypeptide (SEQ ID NO:2). The ORO polypeptidecontains an opossum CH2 IgE domain followed by a rat CH3 IgE domainfollowed by an opossum CH4 IgE domain.

[0033]FIG. 4 is an amino acid sequence listing of an ORO polypeptide(SEQ ID NO:3).

[0034]FIG. 5 is a diagram of the nucleic acid vector designatedpRES-OSO.

[0035]FIG. 6 is a nucleic acid sequence listing of the pRES-OSO vector(SEQ ID NO:4).

[0036]FIG. 7 is a nucleic acid sequence listing of an insert sequencethat encodes an OSO polypeptide (SEQ ID NO:5). The OSO polypeptidecontains an opossum CH2 IgE domain followed by a human CH3 IgE domainfollowed by an opossum CH4 IgE domain.

[0037]FIG. 8 is an amino acid sequence listing of an OSO polypeptide(SEQ ID NO:6).

[0038]FIG. 9 is a nucleic acid sequence listing of an insert sequencethat encodes an ORORO polypeptide (SEQ ID NO:7). The ORORO polypeptidecontains an opossum CH2 IgE domain followed by a rat CH3 IgE domainfollowed by an opossum CH2 IgE domain followed by a rat CH3 IgE domainfollowed by an opossum CH4 IgE domain.

[0039]FIG. 10 is an amino acid sequence listing of an ORORO polypeptide(SEQ ID NO:8).

[0040]FIG. 11 is a nucleic acid sequence listing of an insert sequencethat encodes a modOSOSO-H polypeptide (SEQ ID NO:9). The modOSOSO-Hpolypeptide contains an opossum CH2 IgE domain followed by a human CH3IgE domain followed by an opossum CH2 IgE domain followed by a human CH3IgE domain followed by an opossum CH4 IgE domain. The modOSOSO-Hpolypeptide also contains point mutations in the human CH3 domains thatabolish mast cell receptor binding and a C-terminal polyhistidine tag.

[0041]FIG. 12 is an amino acid sequence listing of a modOSOSO-Hpolypeptide (SEQ ID NO:10).

[0042]FIG. 13 is a nucleic acid sequence listing of an insert sequencethat encodes a modOSOSO polypeptide (SEQ ID NO:11). The modOSOSOpolypeptide contains an opossum CH2 IgE domain followed by a human CH3IgE domain followed by an opossum CH2 IgE domain followed by a human CH3IgE domain followed by an opossum CH4 IgE domain. The modOSOSOpolypeptide also contains point mutations in the human CH3 domains thatabolish mast cell receptor binding.

[0043]FIG. 14 is an amino acid sequence listing of a modOSOSOpolypeptide (SEQ ID NO:12).

[0044]FIG. 15 is a nucleic acid sequence listing of an insert sequencethat encodes an OSO-H polypeptide (SEQ ID NO:13). The OSO-H polypeptidecontains an opossum CH2 IgE domain followed by a human CH3 IgE domainfollowed by an opossum CH4 IgE domain. The OSO-H polypeptide alsocontains a C-terminal polyhistidine tag.

[0045]FIG. 16 is an amino acid sequence listing of an OSO-H polypeptide(SEQ ID NO:14).

[0046]FIG. 17 is a nucleic acid sequence listing of an insert sequencethat encodes an OSOSO polypeptide (SEQ ID NO:15). The OSOSO polypeptidecontains an opossum CH2 IgE domain followed by a human CH3 IgE domainfollowed by an opossum CH2 IgE domain followed by a human CH3 IgE domainfollowed by an opossum CH4 IgE domain.

[0047]FIG. 18 is an amino acid sequence listing of an OSOSO polypeptide(SEQ ID NO:16).

[0048]FIG. 19 is a nucleic acid sequence listing of an insert sequencethat encodes an OSOSO-H polypeptide (SEQ ID NO:17). The OSOSO-Hpolypeptide contains an opossum CH2 IgE domain followed by a human CH3IgE domain followed by an opossum CH2 IgE domain followed by a human CH3IgE domain followed by an opossum CH4 IgE domain. The OSOSO-Hpolypeptide also contains a C-terminal polyhistidine tag.

[0049]FIG. 20 is an amino acid sequence listing of an OSOSO-Hpolypeptide (SEQ ID NO:18).

[0050]FIG. 21 is a nucleic acid sequence listing of an insert sequencethat encodes a CCC-H polypeptide (SEQ ID NO:19). The CCC-H polypeptidecontains a monkey CH2 IgE domain followed by a monkey CH3 IgE domainfollowed by a monkey CH4 IgE domain followed by a polyhistidine tag.

[0051]FIG. 22 is a nucleic acid sequence listing of an insert sequencethat encodes a H-OCO-H polypeptide (SEQ ID NO:20). The H-OCO-Hpolypeptide contains an opossum CH2 IgE domain followed by a monkey CH3IgE domain followed by an opossum CH4 IgE domain. The H-OCO-Hpolypeptide also contains N- and C-terminal polyhistidine tags.

[0052]FIG. 23 is an amino acid sequence listing of an H-OCO-Hpolypeptide (SEQ ID NO:21).

[0053]FIG. 24 is a nucleic acid sequence listing of an insert sequencethat encodes a H-OCOCO-H polypeptide (SEQ ID NO:22). The H-OCOCO-Hpolypeptide contains an opossum CH2 IgE domain followed by a monkey CH3IgE domain followed by an opossum CH2 IgE domain followed by a monkeyCH3 IgE domain followed by an opossum CH4 IgE domain. The H-OCOCO-Hpolypeptide also contains N- and C-terminal polyhistidine tags.

[0054]FIG. 25 is a schematic of an immune response.

[0055]FIG. 26 is a schematic of an IgE molecule.

[0056]FIG. 27 is a schematic of a vaccine having human and opossum IgEsequences.

[0057]FIG. 28 is a schematic of IgE clearance.

[0058]FIG. 29A is a schematic of an H-ORO DNA construct labeling thepositions of the rat and opossum IgE coding sequences. FIG. 29B is aschematic showing the structure of a recombinant H-ORO polypeptide.

[0059]FIG. 30A is a bar graph showing relative anti-rat IgE antibodytiters (anti-self IgE) in rats vaccinated with H-ORO mixed with Freund'sadjuvant, alum, or ISCOM. FIG. 30B is a bar graph showing relativeanti-opossum antibody titers (anti non-self) in the same rats.

[0060]FIG. 31A is a bar graph showing relative anti-rat IgE antibodytiters in rats vaccinated with H-ORO mixed with Freund's adjuvant,MONTANIDE® ISA 51 (MN51), or MONTANIDE® ISA 720 (MN720). FIG. 31B is abar graph showing relative anti-opossum antibody titers in the samerats.

[0061]FIG. 32A is a bar graph showing relative anti-rat IgE antibodytiters in rats vaccinated with H-ORO mixed with MN51, with or withoutthe addition of muramyldipeptide (MDP), monophosphoryl lipid A (MPL),and/or a formyl-methionine containing tripeptide (FM). FIG. 32B is a bargraph showing relative anti-opossum antibody titers in the same rats.

[0062]FIG. 33A is a bar graph showing relative anti-rat IgE antibodytiters in rats vaccinated with H-ORO mixed with MN720, with or withoutthe addition of muramyldipeptide (MDP) and/or monophosphoryl lipid A(MPL). FIG. 33B is a bar graph showing relative anti-opossum antibodytiters in the same rats.

[0063]FIG. 34 is a line graph showing free IgE levels in sera from ratsimmunized with either vehicle mixed with alum or H-ORO mixed with alum.

[0064]FIG. 35A is line graph showing the titer dilution curve for ratanti-IgE antibodies in serum samples from rats immunized with H-OROmixed with MN51. FIG. 35B is a line graph showing the titer dilutioncurve for rat anti-IgE antibodies in serum samples from rats immunizedwith H-ORO mixed with alum. The broken lines represent the 95%confidence interval of the anti-IgE response (n=9−10).

[0065]FIG. 36 is a line graph showing free IgE levels in sera from ratsimmunized with vehicle or ORO-H mixed with Montanide ISA 51.

[0066]FIG. 37 is a line graph showing free IgE levels in sera from ratsimmunized with vehicle or ORORO-H mixed with Montanide ISA 51.

[0067] FIGS. 38A-C show the outline of a study design (A), anti-IgEtiters (B), and free circulating IgE levels (C) in sera from ratsimmunized with vehicle, vehicle mixed with MN51, or increasing amountsof H-ORO mixed with MN51.

[0068]FIG. 39 is a table listing a rat vaccination protocol for a highlypurified (>98% pure) non-histidine tagged ORO polypeptide.

[0069]FIG. 40 is a graph plotting the amount of rat IgE (ng/mL) measuredin rats receiving the indicated treatment.

[0070]FIG. 41 is a graph plotting the percent reduction of freecirculating IgE measured in rats receiving the indicated treatment.

[0071]FIG. 42 is a schematic of a monkey vaccination protocol.

[0072]FIG. 43 is a schematic of an ELISA used to detect monkey anti-IgEantibodies.

[0073]FIG. 44 is a line graph showing the titer dilution₅₀ values inserum samples from cynomolgus monkeys immunized with vehicle mixed withMN51, H-OCO-H mixed with MN51, or H-OCOCO-H mixed with MN5.

[0074]FIG. 45 is a bar graph plotting the platelet counts for theindicated time points.

[0075]FIG. 46 is a listing of the haematological measurements that werefound to be normal.

[0076]FIG. 47 is a schematic protocol of a monkey vaccination protocolusing Alhydrogel™ as adjuvant.

[0077]FIG. 48 is a graph plotting the titer of monkey anti-IgEantibodies for the indicated time points using different doses ofH-OCO-H mixed with Alhydrogel™.

DETAILED DESCRIPTION

[0078] The invention provides methods and materials related to vaccinesagainst self polypeptides. For example, the invention providescompositions containing a polypeptide and an adjuvant. The polypeptidetypically contains self and non-self components, which can result inboth anti self and anti non-self immune responses when administered to amammal. The adjuvant typically is selected to give a relatively highanti-self response, as compared to compositions containing otheradjuvants.

[0079] The term “polypeptide” as used herein refers to a chain of aminoacids, regardless of length or posttranslational modification (e.g.,phosphorylation or glycosylation). For example, in some embodiments, thepolypeptide can be unmodified such that it lacks modifications such asphosphorylation and glycosylation. The polypeptide can contain part orall of a single naturally-occurring polypeptide, or can be a chimericpolypeptide containing amino acid sequences from two or morenaturally-occurring polypeptides. An “adjuvant” is an immunologicalcompound that can enhance an immune response against a particularantigen such as a polypeptide. Typically, the compositions of theinvention are administered to a mammal such that the mammal producesantibodies against the polypeptide component of the administeredcomposition. The mammal can be a mouse, rat, dog, cat, horse, cow, or aprimate such as a human or a non-human primate (e.g., a cynomolgusmonkey).

[0080] In some embodiments, the compositions of the invention can elicitan anti-self polypeptide antibody response in a mammal. For example, apolypeptide can contain one or more self polypeptide segments (e.g., aself polypeptide sequence) with or without one or more non-selfpolypeptide segments (e.g., a non-self polypeptide sequence). The term“self” as used herein with reference to a polypeptide sequence and aparticular mammal refers to a sequence that is seen as self from theprospective of that mammal's immune system. Typically, a selfpolypeptide segment is an amino acid sequence that is identical orsimilar to a sequence from a polypeptide that is native to the speciesof mammal to which the composition is to be administered. The term“non-self” as used herein with reference to a polypeptide sequence and aparticular mammal refers to a sequence that is seen as foreign from theprospective of that mammal's immune system. Typically, a non-selfpolypeptide segment is an amino acid sequence that is not native to thespecies of mammal to which the composition is to be administered. Apolypeptide can be, for example, an ORO polypeptide that containssequences from the rat and opossum IgE molecules and can be administeredto a rat as described herein.

[0081] The polypeptides provided herein can contain more than one copyof the self segment (e.g., an ORORO polypeptide that contains two copiesof a segment from the rat IgE amino acid sequence). Segments frompolypeptides of any type of mammal (e.g., mouse, rat, dog, cat, horse,cow, non-human primate such as cynomolgus monkey, or human) can beincluded in the polypeptides provided herein. For example, any of thepolypeptides described in PCT Application Serial No. PCT/SE99/01896 canbe used. Alternatively, the polypeptides can contain a tag (e.g., a Histag, a myc tag, or a FLAG® tag). Such tags typically are positioned atthe amino terminus or the carboxyl terminus of the polypeptide, but canbe positioned anywhere within the polypeptide. These tags can serve as anon-self component while aiding in the detection and/or purification ofthe polypeptides.

[0082] The self segment or segments, as well as the non-self segment orsegments, can have any length, and typically are at least 5 amino acidsin length (e.g., at least about 5, 10, 20, 30, 40, 50, 60, 65, 70, 75,80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 150, 175, 200, 500, 750,1000, 2000, 3000, 4000, 5000, or more amino acids in length). Forexample, the self segment or segments, as well as the non-self segmentor segments, can have a length ranging from about 20, 30, 40, 50, 60,70, or 80 amino acids to about 90, 100, 110, 120, 130, 140, 150, 200,250, or 500 amino acids. Typically, the self segment (or segments) of apolypeptide has an amino acid sequence that is at least 80 (e.g., 85,90, 95, or 99) percent identical to the amino acid sequence of thepolypeptide that is native to the mammal to which the composition willbe administered. For example, when vaccinating a human, a self IgEsegment of a chimeric IgE polypeptide can be about 110 amino acids inlength with about 95 percent identity to human IgE sequences over that110 amino acid length.

[0083] A length and percent identity over that length for any nucleicacid or amino acid sequence is determined as follows. First, a nucleicacid or amino acid sequence is compared to the identified nucleic acidor amino acid sequence using the BLAST 2 Sequences (Bl2seq) program fromthe stand-alone version of BLASTZ containing BLASTN version 2.0.14 andBLASTP version 2.0.14. This stand-alone version of BLASTZ can beobtained at Fish & Richardson's web site (www.fr.com/blast; World WideWeb at “fr” dot “com” slash “blast”) or the U.S. government's NationalCenter for Biotechnology Information web site(www.ncbi.nlm.nih.gov/blast/executables; World Wide Web at “ncbi” dot“nlm” dot “nih” dot “gov” slash “blast” slash “executables”).Instructions explaining how to use the Bl2seq program can be found inthe readme file accompanying BLASTZ.

[0084] Bl2seq performs a comparison between two sequences using eitherthe BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acidsequences, while BLASTP is used to compare amino acid sequences. Tocompare two nucleic acid sequences, the options are set as follows: −iis set to a file containing the first nucleic acid sequence to becompared (e.g., C:\seq1.txt); −j is set to a file containing the secondnucleic acid sequence to be compared (e.g., C:\seq2.txt); −p is set toblastn; −o is set to any desired file name (e.g., C:\output.txt); −q isset to −1; −r is set to 2; and all other options are left at theirdefault setting. For example, the following command can be used togenerate an output file containing a comparison between two sequences:C:\Bl2seq −i c:\seq1.txt −j c:\seq2.txt −p blastn −o c:\output.txt −q −1−r 2. To compare two amino acid sequences, the options of Bl2seq are setas follows: −i is set to a file containing the first amino acid sequenceto be compared (e.g., C:\seq1.txt); −j is set to a file containing thesecond amino acid sequence to be compared (e.g., C:\seq2.txt); −p is setto blastp; −o is set to any desired file name (e.g., C:\output.txt); andall other options are left at their default setting. For example, thefollowing command can be used to generate an output file containing acomparison between two amino acid sequences: C:\Bl2seq −i c:\seq1.txt −jc:\seq2.txt −p blastp −o c:\output.txt. If the target sequence shareshomology with any portion of the identified sequence, then thedesignated output file will present those regions of homology as alignedsequences. If the target sequence does not share homology with anyportion of the identified sequence, then the designated output file willnot present aligned sequences. Once aligned, a length is determined bycounting the number of consecutive nucleotides or amino acid residuesfrom the target sequence presented in alignment with sequence from theidentified sequence starting with any matched position and ending withany other matched position. A matched position is any position where anidentical nucleotide or amino acid residue is presented in both thetarget and identified sequence. Gaps presented in the target sequenceare not counted since gaps are not nucleotides or amino acid residues.Likewise, gaps presented in the identified sequence are not countedsince target sequence nucleotides or amino acid residues are counted,not nucleotides or amino acid residues from the identified sequence.

[0085] The percent identity over a determined length is determined bycounting the number of matched positions over that length and dividingthat number by the length followed by multiplying the resulting value by100. For example, if (1) a 1000 amino acid target sequence is comparedto a 200 amino acid test sequence, (2) the Bl2seq program presents 200amino acids from the target sequence aligned with a region of the testsequence where the first and last nucleotides of that 200 nucleotideregion are matches, and (3) the number of matches over those 200 alignednucleotides is 180, then the 1000 nucleotide target sequence contains alength of 200 and a percent identity over that length of 90 (i.e.,180/200*100=90).

[0086] It will be appreciated that a single nucleic acid or amino acidtarget sequence that aligns with an identified sequence can have manydifferent lengths with each length having its own percent identity. Forexample, a target sequence containing a 20 nucleotide region that alignswith an identified sequence as follows has many different lengthsincluding those listed in Table 1. 1                  20 TargetAGGTCGTGTACTGTCAGTCA (SEQ ID NO:23) Sequence: | || ||| |||| |||| |Identified ACGTGGTGAACTGCCAGTGA (SEQ ID NO:24) Sequence:

[0087] TABLE I Starting Ending Matched Percent Position Position LengthPositions Identity 1 20 20 15 75.0 1 18 18 14 77.8 1 15 15 11 73.3 6 2015 12 80.0 6 17 12 10 83.3 6 15 10 8 80.0 8 20 13 10 76.9 8 16 9 7 77.8

[0088] It is noted that the percent identity value is rounded to thenearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 is roundeddown to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 is rounded upto 78.2. It is also noted that the length value will always be aninteger.

[0089] Any method can be used to obtain a polypeptide. For example,molecular cloning techniques can be used to prepare a nucleic acidconstruct encoding a polypeptide containing self and non-self segments(e.g., ORO). Such a construct can be expressed in an organism such as E.coli or S. cerevisiae, or in a cell line, for example, and then can bepurified from cellular extracts or from culture supernatants.Alternatively, a polypeptide can be chemically synthesized.

[0090] In particular, nucleic acid vectors can be designed to expresschimeric IgE polypeptides. Examples of such nucleic acid vectorsinclude, without limitation, those set forth in FIGS. 1, 2, 5, and 6. Inaddition, nucleic acid vectors can contain an insert sequence. The term“insert sequence” as used herein refers to a nucleic acid sequence thatis inserted into a nucleic acid vector such that that inserted nucleicacid sequence can be expressed. An insert sequence can be a nucleic acidsequence that encodes a chimeric IgE polypeptide such as a polypeptidehaving the amino acid sequence set forth in FIG. 4, 8, 10, 12, 14, 16,18, 20, or 23. Such nucleic acid sequences can be as set forth in FIG.3, 7, 9, 11, 13, 15, 17, 19, 22, or 24. The term “chimeric IgEpolypeptide” as used herein refers to a polypeptide having a combinationof IgE sequences (e.g., full domains, half domains, or quarter domains)from different species. A chimeric IgE polypeptide typically containsIgE constant heavy (CH) chain domains (e.g., CH1, CH2, CH3, or CH4). Forexample, an insert sequence having the sequence set forth in SEQ ID NO:2can encode an opossum CH2-rat CH3-opossum CH4 (ORO) chimeric IgEpolypeptide (SEQ ID NO:3). Other examples of insert sequences include,without limitation, (1) an insert sequence having the sequence set forthin SEQ ID NO:5 that encodes an opossum CH2-human CH3-opossum CH4 (OSO)chimeric IgE polypeptide (SEQ ID NO:6), (2) an insert sequence havingthe sequence set forth in SEQ ID NO:7 that encodes an opossum CH2-ratCH3-opossum CH2-rat CH3-opossum CH4 (ORORO) chimeric IgE polypeptide(SEQ ID NO:8), and (3) an insert sequence having the sequence set forthin SEQ ID NO:15 that encodes an opossum CH2-human CH3-opossum CH2-humanCH3-opossum CH4 (OSOSO) chimeric IgE polypeptide (SEQ ID NO:16). Inaddition, an insert sequence can have a sequence that encodes any of thepolypeptides disclosed in International Patent Application Serial No.PCT/SE99/01896. In addition to rat and human, IgE sequences (e.g.,domains) from other species can be used in chimeric insert sequences.Such species include, without limitation, dog, cat, horse, pig, cow, andmonkey. For example, an insert sequence including IgE domains fromopossum and monkey (e.g., cynomolgus) can encode an opossumCH2-cynomolgus CH3-opossum CH4 (OCO) chimeric IgE polypeptide. Otherinsert sequences having IgE sequences (e.g., domains) from opossum andmonkey include, without limitation, sequences that encode opossumCH2-cynomolgus CH3-opossum CH4 (OCO-H), where the sequence contains aC-terminal histidine-tag; sequences that encode opossum CH2-cynomolgusCH3-opossum CH2-cynomolgus CH3-opossum CH4 (OCOCO); and sequences thatencode opossum CH2-cynomolgus CH3-opossum CH2-cynomolgus CH3-opossumCH4, where the sequence contains a C-terminal histidine-tag (OCOCO-H).

[0091] An insert sequence can be modified. Such modifications caninclude, without limitation, additions, deletions, substitutions, pointmutations, and combinations thereof. An insert sequence can be modifiedto include a C-terminal polyhistidine sequence to aid in thepurification of the polypeptide encoded by the insert sequence.Polyhistidine sequences used for this purpose have been describedelsewhere (Ford et al., Protein Expr. Purif., 2(2-3):95-107, 1991). Forexample, an insert sequence having the sequence set forth in SEQ IDNO:13 can encode an OSO chimeric IgE polypeptide including a C-terminalpolyhistidine sequence (OSO-H; SEQ ID NO:14). An insert sequence can bemodified to contain point mutations. For example, an insert sequencehaving the sequence set forth in SEQ ID NO:11 can encode an OSOSOchimeric IgE polypeptide containing point mutations in the human CH3domains that abolish mast cell receptor binding (modOSOSO; SEQ IDNO:12). Other examples of modified insert sequences include, withoutlimitation, an insert sequence having the sequence set forth in SEQ IDNO:17 that encodes an OSOSO chimeric IgE polypeptide including aC-terminal polyhistidine sequence (OSOSO-H; SEQ ID NO:18) and an insertsequence having the sequence set forth in SEQ ID NO:9 that encodes anOSOSO chimeric IgE polypeptide including a C-terminal polyhistidinesequence and containing point mutations in the human CH3 domains thatabolish mast cell receptor binding (modOSOSO-H; SEQ ID NO:10).

[0092] A nucleic acid vector also can contain components that affect theexpression of the insert sequence. Examples of such components include,without limitation, promoter, enhancer, leader, and polyadenylationsequences. Such components can be operably linked to the insertsequence. The term “operably linked” as used herein refers to anarrangement where components so described are configured so as toperform their usual function. For example, a nucleic acid vector with aninsert sequence encoding an OSOSO chimeric IgE polypeptide also cancontain a cytomegalovirus (CMV) promoter sequence (see, for example,Thomson et al., Proc. Natl. Acad. Sci. U.S.A., 81(3):659-663, 1984), animmunoglobulin (Ig) leader sequence (see, for example, Neuberger et al.,EMBO J., 2(8):1373-1378, 1983), and a bovine growth hormone (bGH)polyadenylation sequence (see, for example, Goodwin et al., J. BiolChem., 267:16330-16334, 1992). In this case, the components can beoperably linked to the insert sequence such that the CMV promoter candrive the expression of the insert sequence including the Ig leadersequence and bGH polyadenylation sequence, the Ig leader sequence candirect the expressed insert sequence into the lumen of the endoplasmicreticulum in preparation for secretion, and the bGH polyadenylationsequence can stabilize the insert sequence transcript.

[0093] In addition, a nucleic acid vector can contain components thataid in the growth, maintenance, or selection of a host cell containingthe nucleic acid vector. Such components include, without limitation,origins of replication and antibiotic selection markers. For example, anucleic acid vector with a CMV promoter sequence, an Ig leader sequence,an SV40 late polyadenylation sequence, and an insert sequence encodingan OSOSO chimeric IgE polypeptide can also contain an f1 origin ofreplication sequence, a sequence that confers ampicillin resistance on abacterial host cell when expressed, and a sequence that confers neomycinresistance on a mammalian host cell when expressed. Other examples ofantibiotic selection markers include, without limitation, sequences thatconfer resistance to hygromycin B, puromycin, kanamycin, tetracycline,blasticidin S, Geneticin®, and zeocin on a host cell when expressed.Nucleic acid vectors that contain one or more than one componentdescribed herein can be obtained commercially from, for example,Invitrogen (Carlsbad, Calif.) and Promega (Madison, Wis.).

[0094] Polypeptide containing self IgE sequences can be obtained usinghost cells containing a nucleic acid vector (e.g., the pCI-neo vectorfrom Promega, catalogue number E1841) with at least one of the insertsequences provided herein (e.g., ORO, OSO, ORORO, modORORO-H, modOSOSO,OSO-H, OSOSO, and OSOSO-H). Such cells can be prokaryotic cells (e.g.,JM109 or DH5α cells) or eukaryotic cells (e.g., NS0, HeLa, BHK-21,COS-7, Sf9, or CHO cells). Host cells containing the nucleic acid vectormay or may not express the encoded polypeptide. For example, a host cellmay function simply to propagate the nucleic acid vector for use inother host cells. In addition, the nucleic acid vector can be integratedinto the genome of the host or maintained in an episomal state. Thus, ahost cell can be stably or transiently transfected with the nucleic acidvector.

[0095] A host cell can contain a nucleic acid vector with an insertsequence that encodes a chimeric IgE polypeptide. For example, a hostcell can contain a nucleic acid vector with an insert sequence encodingan OSO chimeric IgE polypeptide or any of the chimeric IgE polypeptidesprovided herein. In addition, a host cell can express the polypeptideencoded by the insert sequence.

[0096] Various methods can be used to introduce a nucleic acid vectorinto a host cell in vivo or in vitro. For example, calcium phosphateprecipitation, electroporation, heat shock, lipofection, microinjection,and viral-mediated nucleic acid transfer are common methods that can beused to introduce a nucleic acid vector into a host cell. In addition,naked DNA can be delivered directly to cells in vivo as describedelsewhere (U.S. Pat. Nos. 5,580,859 and 5,589,466). Further, a nucleicacid vector can be introduced into cells to generate transgenic animals.

[0097] Transgenic animals can be aquatic animals (such as fish, sharks,dolphin, and the like), farm animals (such as pigs, goats, sheep, cows,horses, rabbits, and the like), rodents (such as rats, guinea pigs, andmice), non-human primates (such as baboon, monkeys, and chimpanzees),and domestic animals (such as dogs and cats). Several techniques knownin the art can be used to introduce a nucleic acid vector into animalsto produce the founder lines of transgenic animals. Such techniquesinclude, without limitation, pronuclear microinjection (U.S. Pat. No.4,873,191); retrovirus mediated gene transfer into germ lines (Van derPutten et al., Proc. Natl. Acad. Sci., USA, 82:6148 (1985)); genetransfection into embryonic stem cells (Gossler A et al, Proc Natl AcadSci USA 83:9065-9069 (1986)); gene targeting into embryonic stem cells(Thompson et al., Cell, 56:313 (1989)); nuclear transfer of somaticnuclei (Schnieke AE et al., Science 278:2130-2133 (1997)); andelectroporation of embryos (Lo CW, Mol. Cell. Biol., 3:1803-1814(1983)). Once obtained, transgenic animals can be replicated usingtraditional breeding or animal cloning.

[0098] Various methods can be used to identify a host cell containing anucleic acid vector provided herein. Such methods include, withoutlimitation, PCR, nucleic acid hybridization techniques such as Northernand Southern analysis, and in situ nucleic acid hybridization. In somecases, immunohistochemistry and biochemical techniques can be used todetermine if a cell contains a nucleic acid vector with a particularinsert sequence by detecting the expression of a polypeptide encoded bythat particular insert sequence.

[0099] Any method can be used to produce recombinant chimeric IgEpolypeptides. Such methods involve culturing a host cell that expressesa chimeric IgE polypeptide and recovering the expressed chimeric IgEpolypeptides. Any method can be used to recover a recombinant chimericIgE polypeptide. For example, recombinant chimeric IgE polypeptides thatare present in a host cell homogenate can be recovered using ionexchange chromatography. In another example, recombinant chimeric IgEpolypeptides with polyhistidine sequences can be recovered from a hostcell homogenate by passing the homogenate over a nickel column andeluting the polyhistidine-containing polypeptides with imidazole. Aparticular recombinant chimeric IgE polypeptide with a leader sequencethat directs that polypeptide's secretion can be recovered from thegrowth medium of a host cell expressing that polypeptide. For example,the growth medium from a culture of mammalian host cells expressing andsecreting ORO or OSO polypeptides can be collected, and the ORO or OSOpolypeptides can be recovered using chromatography. It is understoodthat a leader sequence that directs the secretion of a polypeptidetypically is removed from that polypeptide in the host cell byproteolysis. Thus, the recovered secreted polypeptide, in many cases, isfree of any translated leader sequence.

[0100] In one embodiment, the cell medium from a clonal CHO cell lineexpressing and secreting ORO or OSO polypeptides is collected andcentrifuged to remove cell debris. After centrifuging, the supernatantis dialyzed and passed over an ion exchange column allowing the ORO orOSO polypeptides to bind. The bound ORO or OSO polypeptides are elutedusing a sodium chloride/sodium acetate gradient, and the elutedfractions are screened for recombinant ORO or OSO polypeptides using anELISA technique. The eluted fractions with high ELISA reactivity can bepooled and dialyzed again, and the dialyzed pooled fractions can bepassed over a hydrophobic interaction column allowing the ORO or OSOpolypeptides to bind. The bound ORO or OSO polypeptides are eluted usinga sodium phosphate gradient, and the eluted fractions are again screenedfor recombinant ORO or OSO polypeptides using an ELISA technique. Theeluted fractions with high ELISA reactivity can be further analyzed bysilver stained SDS-PAGE to estimate the purity of the ORO or OSOpolypeptides.

[0101] As described herein, alum as well as other aluminum-basedcompounds (e.g., Al₂O₃) can be combined with a polypeptide containing aself polypeptide segment (e.g., a self IgE sequence) to form acomposition that elicits an anti-self response when administered to amammal. Aluminum-based compounds can be obtained from various commercialsuppliers. For example, REHYDRAGEL® adjuvants can be obtained fromReheis Inc. (Berkeley Heights, N.J.). REHYDRAGEL® adjuvants are based oncrystalline aluminum oxyhydroxide, and are hydrated gels containingcrystalline particles with a large surface area (about 525 m²/g). TheirAl₂O₃ content typically ranges from about 2 percent to about 10 percent.Rehydragel LG, for example, has an Al₂O₃ content of about 6 percent, andflows readily upon slight agitation. Rehydragel LG also has a proteinbinding capacity of 1.58 (i.e., 1.58 mg of bovine serum albumin boundper 1 mg of Al₂O₃), a sodium content of 0.02 percent, a chloride contentof 0.28 percent, undetectable sulphate, an arsenic level less than 3ppm, a heavy metal content less than 15 ppm, a pH of 6.5, and aviscosity of 1090 cp. Rehydragel LG can be combined with a polypeptidesolution (e.g., a polypeptide in PBS) to yield Al(OH)₃. In addition,ALHYDROGEL™, an aluminum hydroxy gel adjuvant, (Alhydrogel 1.3%,Alhydrogel 2.0%, or Alhydrogel “85”) obtained from Brenntag StinnesLogistics can be used.

[0102] In addition, MN51 can be combined with a polypeptide containing aself polypeptide segment (e.g., a self IgE sequence) to form acomposition that elicits an anti-self response when administered to amammal. MN51 (MONTANIDE® Incomplete SEPPIC Adjuvant (ISA) 51) as well asMN720 are available from Seppic (Paris, France). MN51 contains mannideoleate (MONTANIDE® 80, also known as anhydro mannitol octadecenoate) inmineral oil solution (Drakeol 6 VR). MONTANIDE® 80 is a limpid liquidwith a maximum acid value of 1, a saponification value of 164-172, ahydroxyl value of 89-100, an iodine value of 67-75, a maximum peroxidevalue of 2, a heavy metal value less than 20 ppm, a maximum watercontent of 0.35%, a maximum color value of 9, and a viscosity at 25° C.of about 300 mPas. MONTANIDE® associated with oil (e.g., mineral oil,vegetable oil, squalane, squalene, or esters) is known as MONTANIDE®ISA. Drakeol 6 VR is a pharmaceutical grade mineral oil. Drakeol 6 VRcontains no unsaturated or aromatic hydrocarbons, and has an A.P.I.gravity of 36.2-36.8, a specific gravity at 25° C. of 0.834-0.838, aviscosity at 100° F. of 59-61 SSU or 10.0-10.6 centistokes, a refractiveindex at 25° C. of 1.458-1.463, a better than minimum acid test, isnegative for fluorescence at 360 nm, is negative for visible suspendedmatter, has an ASTM pour test value of 0-15° F., has a minimum ASTMflash point of 295° F., and complies with all RN requirements for lightmineral oil and ultraviolet absorption. MN51 contains about 8 to 12percent anhydro mannitol octadecenoate and about 88 to 92 percentmineral oil. MN51 is a clear yellow liquid having a maximum acid valueof 0.5, a saponification value of 16-20, a hydroxyl value of 9-13, amaximum peroxide value of 2, an iodine value of 5-9, a maximum watercontent of 0.5 percent, a refractive index at 25° C. between 1.455 and1.465, a density at 20° C. of about 0.85, and a viscosity at 20° C. ofabout 50 mPaS. The conductivity of a 50:50 mixture of MN51 and saline isless than 10 μScm⁻¹.

[0103] Other adjuvants include immuno-stimulating complexes (ISCOMs)that can contain such components as cholesterol and saponins. ISCOMmatrices can be prepared and conjugated to Cu²⁺ using methods such asthose described herein. Adjuvants such as FCA, FIA, MN51, MN720, andAl(OH)₃ are commercially available from companies such as Seppic, DifcoLaboratories (Detroit, Mich.), and Superfos Biosector A/S (Vedbeak,Demark).

[0104] In some embodiments, a composition also can contain one or moreadditional immunostimulatory components. These include, withoutlimitation, muramyldipeptide (e.g.,N-acetylmuramyl-L-alanyl-D-isoglutamine; MDP), monophosphoryl-lipid A(MPL), and formyl-methionine containing tripeptides such asN-formyl-Met-Leu-Phe. Such compounds are commercially available fromSigma Chemical Co. (St. Louis, Mo.) and RIBI ImmunoChem Research, Inc.(Hamilton, Mont.), for example.

[0105] A “unit dose” of a composition refers to the amount of acomposition administered to a mammal at one time. A unit dose of thecompositions provided herein can contain any amount of polypeptide. Forexample, a unit dose of a composition can contain between about 10 μgand about 1 g (e.g., 10 μg, 15 μg, 25 μg, 30 μg, 50 μg, 100 μg, 250 μg,280 μg, 300 μg, 500 μg, 750 μg, 1 mg, 10 mg, 15 mg, 25 mg, 50 mg, 50 mg,100 mg, 250 mg, 280 mg, 300 mg, 500 mg, 750 mg, or more) of apolypeptide. In some embodiments, the polypeptide can be dissolved orsuspended in a physiological buffer such as, for example, water orphosphate buffered saline (PBS), pH 7.0. The solution of polypeptidethen can be combined with the adjuvant and any other components of thecomposition.

[0106] Similarly, a unit dose of a composition can contain any amount ofan adjuvant. For example, a unit dose can contain between about 10 μLand about 1 mL (e.g., 10 μL, 25 μL, 50 μL, 100 μL, 250 μL, 500 μL, 750μL, 800 μL, 900 μL, or 1 mL) of one or more adjuvants. In addition, aunit dose of a composition can contain any amount of anotherimmunostimulatory component. For example, a composition provided hereincan contain between about 10 μg and about 1 g (e.g., 10 μg, 15 μg, 25μg, 30 μg, 50 μg, 100 μg, 250 μg, 280 μg, 300 μg, 500 μg, 750 μg, 1 mg,10 mg, 15 mg, 25 mg, 30 mg, 50 mg, 100 mg, 250 mg, 280 mg, 300 mg, 500mg, 750 mg, or more) of an immunostimulatory component.

[0107] The compositions provided herein can contain any ratio ofadjuvant to polypeptide. The adjuvant:antigen ratio can be 50:50(vol:vol), for example. Alternatively, the adjuvant:antigen ratio canbe, without limitation, 90:10, 80:20, 70:30, 64:36, 60:40, 55:45, 40:60,30:70, 20:80, or 90:10.

[0108] The invention also provides methods for preparing thecompositions provided herein. Such methods can involve suspending anamount of a polypeptide (e.g., 100 μg of ORO) in a suitable amount of aphysiological buffer (e.g., 50 μL of PBS pH 7.0), and then combining thesuspended or dissolved antigen with a suitable amount of an adjuvant(e.g., 50 μL of MN51 or 100 μL of REHYDRAGEL®). The combining step canbe achieved by any method, including stirring, shaking, vortexing, orpassing back and forth through a needle attached to a syringe, forexample. It is noted that the composition can be prepared in batch, suchthat enough unit doses are obtained for multiple injections (e.g.,injections into multiple animals or multiple injections into the sameanimal).

[0109] The invention also provides methods for inducing an anti-selfresponse in a mammal (e.g., a mouse, a rat, a cat, a dog, a horse, acow, a non-human primate such as a cynomolgus monkey, or a human). Suchmethods can involve administering to a mammal a composition providedherein, wherein the composition contains a polypeptide that includes anamino acid sequence from a self polypeptide (e.g., an amino acidsequence from the CH3 domain of an IgE polypeptide found in thatparticular species of mammal). The polypeptide can contain at least oneamino acid sequence from another species (e.g., an amino acid sequencefrom the CH2 or CH4 domain of an IgE polypeptide found in a differentspecies).

[0110] In general, compositions containing a polypeptide provided hereincan be used as an allergy vaccine to abrogate the allergic cascade byeliminating circulating IgE (FIGS. 25-28). The compositions can inducean antibody response against self-IgE in the recipient. Although notlimited to any particular mode of action, it is believed thatadministration of compositions containing a polypeptide with self IgEsequences in a context which allows the mammal's tolerance to IgE to bebroken leads to the production of anti-self IgE antibodies, which inturn decreases the level of circulating self IgE antibodies.

[0111] The compositions provided herein can be administered by a numberof methods. Administration can be, for example, topical (e.g.,transdermal, ophthalmic, or intranasal); pulmonary (e.g., by inhalationor insufflation of powders or aerosols); oral; or parenteral (e.g., bysubcutaneous, intrathecal, intraventricular, intramuscular, orintraperitoneal injection, or by intravenous drip). Administration canbe rapid (e.g., by injection) or can occur over a period of time (e.g.,by slow infusion or administration of slow release formulations).

[0112] Any dose can be administered to a mammal. Dosages can varydepending on the relative potency of individual compositions, and cangenerally be estimated based on data obtained from in vitro and in vivoanimal models. Typically, dosage is from about 0.01 μg to about 100 gper kg of body weight, and may be given once or more daily, weekly, oreven less often. Following successful administration, it may bedesirable to have the subject undergo additional booster administrationsto maintain a suitable level of the anti-self response.

[0113] The anti-self response (e.g., anti-self IgE antibody response) toa composition in a mammal can be assessed using any method. For example,the anti-self IgE titer can be measured. Alternatively, a “titerdilution₅₀ value” can be determined by using an ELISA and measuring theoptical density (OD) of dilutions (e.g., serial dilutions) of the serumsamples. The dilution factor that results in a 50 percent reduction fromthe maximal OD is considered to be the titer dilution₅₀ value. Thisvalue can be calculated by curve fitting using, for example, theSOFTmax® Pro 4.0 software program that is available from MolecularDevices, Inc. (Sunnyvale, Calif.). Using a four parameter non-linearregression for curve fitting, this program can be used to fit datapoints to a curve and determine the titer dilution₅₀ value.

[0114] The invention also provides methods for measuring free IgE levelsin the serum of a subject (e.g., a mammal) treated with a polypeptidecontaining one or more self IgE segments (e.g., ORO). Such methods caninvolve providing a serum sample from a subject treated with, forexample, ORO, and incubating the sample with an IgE receptor polypeptidesuch as the human IgE receptor alpha-chain (e.g., the polypeptide havingGenBank® Accession No. NM_(—)002001) to form IgE/IgE receptor complexes.Any IgE receptor sequence (or portion thereof) can be used. For example,a human IgE receptor alpha-chain can be used to measure free IgE inhumans or other primates such as monkeys. After incubating the IgEreceptor polypeptide with the sample containing free IgE, the formedIgE/IgE receptor complexes can be measured. Any method can be used tomeasure IgE/IgE receptor complexes. For example, immunological assayssuch as ELISAs and ELISA-like procedures can be used to measure IgE/IgEreceptor complexes.

[0115] The invention also provides kits for assessing the amount of freeIgE present in a mammal treated with an anti-self IgEpolypeptide-containing composition. Such kits can contain an IgEreceptor sequence and an antibody capable of binding to an IgE/IgEreceptor complex. The kits provided herein also can contain acomposition described herein such as an ORO-containing composition. Suchkits can be used to assess free IgE levels in a mammal and, if needed,to provide an additional booster of the self polypeptide-containingcomposition. The kits provided herein can contain additional reagentssuch as IgE standards, negative controls, enzyme preparations, andenzyme substrates.

[0116] The invention will be further described in the followingexamples, which do not limit the scope of the invention described in theclaims.

EXAMPLES Example 1 Evaluation of Vaccine Adjuvants

[0117] Production of the Active Vaccine Component, H-ORO

[0118] The active component in the vaccine, H-ORO, was encoded by arecombinant construct containing 1041 bp from the C3 domain of ratε-heavy chain (Hellman et al., Nucl. Acids Res., 10:6041 (1982)) flankedby the C2 and C4 domains of the opossum ε-heavy chain (Aveskogh andHellman, Eur. J. Immunol., 28:2738 (1998)). This construct (FIG. 29) wasexpressed in 293-EBNA cells and purified on Ni-NTA Agarose (QIAGEN GmbH,Germany) as described previously (Vemersson et al., FASEB J., 16:875(2002)). The H-ORO component was obtained at a concentration of 1.5mg/mL in PBS pH 7.0.

[0119] Study 1:

[0120] Twenty, 8-10 week old female Wistar rats (Benton and Kingman,Sollentuna, Sweden) were sensitized against ovalbumin (OVA). The animalsreceived an initial intraperitoneal (i.p.) injection of 10 μg OVA (SigmaChemical Co., MO) in PBS pH 7.0, followed by weekly i.p. injections of 3μg OVA in PBS pH 7.0 for 5 weeks prior to the initial vaccination, andcontinuing throughout the vaccination program.

[0121] Five groups of four animals received an initial i.p. vaccinationof H-ORO mixed with one of the following adjuvants: FCA (DifcoLaboratories, Detroit, Mich.), Al(OH)₃ (Superfos Biosector A/S, Vedbeak,Denmark), or Cu²⁺-conjugated ISCOM matrix (Andersson et al. (2001) J.Immunol. Methods 255:135). Animals 1-4 received 100 μg H-ORO in 50 μLPBS pH 7.0, mixed 50:50 with FCA. Animals 5-8 received 100 μg H-ORO and10 vol % Al(OH)₃ slurry in 100 μL PBS pH 7.0. Animals 9-12 received 100μg H-ORO in 50 μL PBS pH 7.0, mixed 50:50 with Cu²⁺-conjugated ISCOMmatrix. Animals 13-16 received 25 μg H-ORO in 50 μL PBS pH 7.0, mixed50:50 with Cu²⁺-conjugated ISCOM matrix. Booster vaccinations wereadministered in week three of the treatment program. The boostervaccinations were identical to the initial vaccinations, with theexception that FIA (Difco Laboratories) was used instead of FCA inanimals 1-4.

[0122] Blood samples of 1 mL were collected from the tail vein beforeinitiating sensitization, three days prior to vaccination, and two weeksafter the booster vaccination. The blood was allowed to coagulateovernight at 4° C. and spun down for 10 minutes at 10,000 rpm (EBA12R,Hettich Zentrifugen, Germany). The sera were transferred to Eppendorftubes and frozen until evaluation by ELISA.

[0123] The vaccine preparations containing Al(OH)₃ were mixed to a 10vol % Al(OH)₃ slurry with 100 μg H-ORO protein in PBS pH 7.0 one dayprior to vaccination, and stored at 4° C. overnight. The ISCOM matrixwas prepared as follows: IDA Matrix with Cu²⁺ had an estimated QAcontent of 1.7 mg/mL and an estimated cholesterol content of 0.5 mg/mL(Prep. 990823 B). Matrix without Cu²⁺ had a QA content of 2.6 mg/mL, andcholesterol was estimated to be 0.8 mg/mL (Prep. 990320). To load thematrix with Cu²⁺; a stock solution of 1 M CuSO₄*5H₂0 in water wasprepared. This solution was added to the matrix preparation to a finalconcentration of 0.1 M Cu²⁺. The mixture was incubated on a shaker inroom temperature for 30 minutes, followed by dialysis against PBSovernight at 4° C. Protein antigen was added in a ratio of 1:1 to thecholesterol content and incubated at 4° C. overnight.

[0124] Study 2:

[0125] A total of forty, 8-10 weeks old female Wistar rats divided intoten groups of four animals received an initial 200 μL i.p. injection of100 μg H-ORO in PBS pH 7.0, together with an adjuvant and in somegroups, additional immunostimulators. Animals 1-4 received FCA mixed ata 50:50 ratio with antigen. Animals 5-8 received MN51 (Seppic, ParisCedex 07, France) at a 50:50 ratio to the antigen. Animals 9-12 wereinjected with MN51:antigen (50:50) and 200 μg MDP (Sigma Chemical Co.)per animal (25 μg of MDP was dissolved in sterile PBS to a finalconcentration of 10 mg/mL). Group 4, animals 13-16, receivedMN51:antigen (50:50) and 200 μg MPL. A 10 mg/mL solution of MPL (RIBIImmunoChem Research, Inc., Hamilton, Mont.) in methanol/chloroform (1:4)was aliquoted in volumes corresponding to doses of 0.2 mg and 0.1 mg MPLper animal and evaporated. Animals 17-20 were given MN51:antigen(50:50), 200 μg MDP, and 100 μg MPL. Animals 21-24 received MN51:antigen(50:50), 200 μg MDP, 100 μg MPL, and 100 μg fMLP (Sigma Chemical Co.).Ten mg of fMLP was dissolved in 1 mL sterile PBS and 1 mL 95% ethanol.Animals 25-28 received MN720 (Seppic) in a 70:30 ratio with H-ORO.Animals 29-32 were injected with MN720:antigen (64:36) and 200 μg MDPper animal. Animals 33-36 were injected with MN720:antigen (70:30) and200 μg MPL. Group 10, animals 37-40, were given MN720:antigen (64:36)with the addition of 200 μg MDP and 100 μg MPL.

[0126] The booster dose contained half the amount of H-ORO (50 μg) givenin the initial vaccination, and FIA was used instead of FCA. Blood wasdrawn from the tail vein ten days prior to vaccination and two weeksafter the booster. The blood was treated as described in Study 1.

[0127] Anti Rat-IgE ELISA:

[0128] The anti rat-IgE ELISA has been described previously (Vernerssonet al., supra). The samples were assayed in singles and horse seraserved as the assay blank. In order to correlate the values, serialdilutions of two of the samples were assayed on every plate.

[0129] Anti Opossum C2-C3-C4 ELISA:

[0130] The same procedure as above (Vernersson et al., supra) was used,except for the coating antigen, which in this case was opossum C2C3C4 ata concentration of 5 μg/mL in carbonate buffer pH 9.6.

[0131] The H-ORO Component:

[0132] To address the question of difference in immune response againstself and non-self components, a hybrid molecule containing both self andnon-self regions was designed and produced as a recombinant protein. Avaccine containing the third constant domain from the rat ε-heavy chain(the target species) flanked by the second and fourth constant domainsof the American opossum IgE heavy chain (OpossumCH2-RatCH3-OpossumCH4;H-ORO, FIG. 29) was expressed in 293-EBNA human embryonic kidney cells.The average yield was about 1 mg H-ORO protein per liter conditionedmedia. Based on SDS-PAGE, the purity of H-ORO was estimated to be atleast 90%, and the major contaminant was identified as BSA derived fromthe FBS-supplemented cell culture medium (Vernersson et al., supra). Theopossum sequences differed in sequence by almost 60% from rat IgE, andthereby served as a non-self component. The opossum domains had twoadditional functions, acting both as structural support for theself-component (the C3 domain) and to break T cell tolerance to the selfcomponent by providing foreign T cell epitopes.

[0133] As a reagent for measuring anti opossum responses by ELISA, arecombinant opossum C2C3C4 IgE was produced (OOO) by the same procedureas described above. Purified whole rat IgE was used for measurements ofthe anti rat-IgE C3 responses.

[0134] Results:

[0135] Three adjuvants were studied: Freund's adjuvant, Alum (Al(OH)₃),and a preparation of ISCOMs. The various adjuvants were administered byi.p. injection together with the H-ORO vaccine component. The animalswere divided into four groups: four animals were given 100 μg of H-OROin CFA, four animals received the same amount of protein absorbed toAlum (a 10 vol % Al(OH)₃ slurry) from a commercially availablepreparation, four animals received 100 μg of H-ORO absorbed to thesurface of 100 μg of ISCOMs, and the last four animals were administeredthe same amount of ISCOMs but only 25 μg of H-ORO. The rationale behindthe reduced levels of antigen was to study the effect of a lower loadingdensity on the ISCOMs, which may influence the availability for theimmune system to recognize the surface epitopes.

[0136] Booster vaccinations were administered in week three of thetreatment program. The booster vaccinations were identical to theinitial vaccinations, with the exception that IFA was used instead ofFCA in animals 1-4. Blood samples of 1 mL were collected from the tailvein three days prior to vaccination and two weeks after the boostervaccination.

[0137] Comparative ELISA analyses were performed on sera from week 5 ofthe treatment program. The ELISA plates were either coated with wholerat IgE in order to measure anti rat C3 immune responses (theanti-self-response), or with opossum C2C3C4 recombinant protein (OOO) tomeasure anti non-self responses. Surprisingly, substantialanti-self-responses were detected only with Freund's adjuvant (FIG.30A). No response was detected with Alum in this experiment, and aresponse was observed only in one of the four animals that received the25 μg dose of H-ORO absorbed on ISCOMs. However, when measuring the antinon-self responses, the ISCOMs were comparable with Freund's, and nosignificant difference in magnitude between these two adjuvants could bedetected. However, Alum was shown to be a less potent adjuvant. Alumgave a response of approximately 20% of the levels seen with Freund'sand the ISCOMs (FIG. 30B).

[0138] The relative difference between the self and the non-selfresponses also was estimated by performing an ELISA assay in whichdifferent wells on the same plate were coated with either rat IgE orOOO. The ratios between anti non-self response and theanti-self-response in the Freund's treated animals were found to be 150,175, 150 and 750 for the four animals, giving a mean value ofapproximately 300 times difference in titer. Although a substantialinduction of anti-self-antibodies was observed, this suggests that thetiters of antibodies against self IgE sequences were substantially lowerthan the titers of antibodies against the non-self IgE sequences.

[0139] Although the induction of anti non-self immune responses with theISCOM preparation was comparable in magnitude to the levels obtained inthe animals given Freund's adjuvant, the anti-self-IgE titers wereundetectable or very low. In addition, no detectable anti-self IgE wasdetected with the Alum preparation. The only adjuvant that resulted insignificant levels of anti-self IgE antibodies was Freund's adjuvant, anadjuvant based on mineral oil.

Example 2 A Comparative Analysis of MN51 and MN720

[0140] One commercially available mineral oil adjuvant (MN51) and oneadjuvant (MN720) that is based on plant oil but has the same emulsifier(mannide monooleate) as MN51 were tested to compare their effects withthe effects of Freund's adjuvant.

[0141] As in the first experiment, four animals in each group weretested for the induction of anti-self and anti non-self responses. Theamounts of antigen and adjuvant were 100 μL of adjuvant and 100 μg ofH-ORO. Comparative ELISA analyses were performed using sera obtained inweek 5 of the treatment program. A substantial anti-self IgE responsewas detected in all animals. The most prominent response, however, wasseen with MN51, which actually was slightly higher (130%) than theresponse observed with Freund's adjuvant (FIG. 31A). MN720 produced aresponse corresponding to only about 15% of the response seen withFreund's. In contrast to the observation for the anti-self-response,however, all three adjuvants were almost equally potent in theirabilities to induce an anti non-self response (FIG. 31B).

[0142] The relative magnitudes of the anti-non-self and anti-selfresponses also were determined. The ratio between the anti non-selfresponse and the anti-self-response in the Freund's treated animals was50, 65, and 75 for three of the four animals, giving a mean value ofabout 63 times difference in titer. The amount of sera obtained from thefourth animal was insufficient to conduct this analysis. For MN51, theratios were 200, 30, 40, and 26 for the four animals, giving a meanvalue of 74 times difference in titer.

Example 3 An Analysis of Potential Additive Effects with MDP, Lipid A,and Formyl-Met Polypeptides

[0143] Based on the results from the two previous experiments, it wasconcluded that the mineral oil based adjuvants are the most effective ininducing anti-self-IgE responses. The difference between anti-self andanti non-self responses can still be quite substantial, however, andprobably frequently exceeds a 50-fold difference. Bacterialimmunostimulatory substances thus were tested in order to determinewhether an additive effect could be achieved. A series of experimentswere conducted in which rats were immunized in groups of four with 100μg H-ORO in MN51 or MN720, with addition of either 200 μg of MDP, 200 μgof MPL, 200 μg of MDP and 100 μg of MPL, or 200 μg of MDP, 100 μg ofMPL, and 100 μg of fMLP.

[0144] Comparative ELISA analyses were performed on sera from week 5 ofthe treatment program. Absorbances were measured, and the values werecompared to a relative absorbance with Freund's set at 100%. Theaddition of MLP, MPL, fMLP, or a combination of two or three of thesedid not have any significant positive effect on the response againsteither the self nor the non-self epitopes when administrated togetherwith MN51 (FIGS. 32A and B). A slight negative effect on theanti-self-response was observed with several of these additions (FIG.32A). In the experiment with MN720, a minor enhancement of theanti-self-response was seen with MPL (FIG. 33A). All additions had aminor negative effect on the anti non-self response, however (FIG. 33B).

Example 4 An Analysis of ORO with Alum

[0145] The effectiveness of alum was further tested in another study, inwhich the alum was prepared from Rehydragel LG. After four weeks ofsensitization with OVA, groups of 9 or 10 female Wistar rats weresubcutaneously immunized with compositions containing vehicle (PBS) withalum, 100 μg H-ORO with MN51, or 280 μg H-ORO with alum. Boosterimmunizations were given at weeks 3 and 7. Serum samples were obtainedat weeks −4, −1, 9, 12, and 15. The samples from week 12 were analyzedfor titer dilution, while all samples were analyzed for free IgEconcentrations using standard methods. The concentration of free IgEdiminished over time in the sera of animals injected with either vehicleplus alum or H-ORO plus alum (FIG. 34). However, the decrease wasgreater in the animals treated with H-ORO plus alum, and theconcentrations of free IgE after 9 weeks were significantly differentbetween the two groups (p<0.01). Immunization with 280 μg H-ORO and alumresulted in a dilution curve that was very similar to that displayed bysera from animals immunized with 100 μg H-ORO and MN51 (FIGS. 35A and35B). In fact, the titer dilutions₅₀ values were calculated to be400-fold for ORO with alum and 204-fold for H-ORO with MN51. Theseresults demonstrate that a composition containing greater than 100 μgH-ORO in combination with alum can induce substantial anti-self IgEresponses when administered to a mammal.

Example 5

[0146] Analysis of ORO-H and ORORO-H

[0147] Similar studies were conducted to evaluate the effects ofcompositions containing ORO and ORORO linked to a His tag. The ORORO-Hpolypeptide contains the following IgE domains:OpossumCH2-RatCH3-OpossumCH2-RatCH3-OpossumCH4. After four weeks ofsensitization with OVA, groups of 6 male Wistar F rats weresubcutaneously immunized with compositions containing vehicle (PBS), 20μg ORO-H, or 100 μg ORO-H. Booster immunizations were given at weeks 3and 7. In each case, Montanide ISA 51 was used as an adjuvant. Serumsamples were obtained at weeks −4, −1, 5, 7, 9, 11, and 14, and wereanalyzed for free IgE concentrations. As shown in FIG. 36, immunizationwith either 20 μg ORO-H or 100 μg ORO-H was equally effective atreducing the concentration of free IgE, while the vehicle resulted in anincrease in free IgE levels.

[0148] In another experiment, groups of 6 or 7 male Wistar rats receivedeither PBS vehicle, 20 μg ORORO-H, or 100 μg ORORO-H via subcutaneousinjection. Booster immunizations were given at weeks 3 and 7, and serumsamples were obtained at weeks −4, −1, 5, 7, 9, 11, and 14. In eachcase, Montanide ISA 51 was used as an adjuvant. As in the experimentdescribed in the paragraph immediately above, immunization with either20 or 100 μg of ORORO-H was highly effective at reducing theconcentration of free IgE, while the vehicle resulted in an increase infree IgE levels (FIG. 37).

Example 6 Dose and Toxicity Studies in Rat

[0149] Groups of 10 male Wister Hanover rats were subcutaneouslyimmunized with vehicle (PBS), vehicle with MN51, 30 μg H-ORO with MN51,100 μg H-ORO with MN51, or 300 μg H-ORO with MN51. All mixtures withMN51 were in a 1:1 ratio. Booster immunizations were given at weeks 1,3, and 5, and blood samples were obtained at weeks 0, 4, and 7 (FIG.38A). The median titer dilution₅₀ values at week 7 increased as the doseof H-ORO increased (FIG. 38B). Thus, higher doses of H-ORO administeredwith MN51 can result in a greater anti-self IgE effect. In addition,free IgE antibody levels were reduced in animals receiving either 100 or300 μg of H-ORO in MN51 (FIG. 38C).

[0150] Toxicity and general health studies also were conducted usingthese animals. Blood samples were evaluated for albumin, ASAT and ALAT,bilirubin, creatinine, electrolytes such as Ca²⁺, K⁺, and Na⁺, lactatedehydrogenase, γ-glutamyl transpeptidase, and glucose, as well ashaemoglobin, white blood cells, hematocrit, and platelet count. Inaddition, the animals were monitored twice weekly for body weight, oncedaily for changes in food intake, and for general physical activity,behavior, and appearance. Furthermore, histopathology studies wereconducted on brain, lungs, ileum, liver, heart, spleen, kidneys, andtesticles. In all of these examinations, no signs of toxic or unwantedeffects were observed.

[0151] In another experiment, groups of 10 female Wister F rats weresubcutaneously immunized with vehicle (PBS) or ORO lacking a histidinetag (FIG. 39). The adjuvant was either MN51 or Alhydrogel™ 1.3% (analuminum hydroxide gel adjuvant; Brenntag Stinnes Logistics). Boosterimmunizations were given at weeks 3, 5, and 7, and blood samples wereobtained at weeks −1, 3, 5, 7, 10, and 12. The amount of rat IgEantibodies measured in rats receiving the ORO polypeptide weresignificantly reduced as compared to the amounts measured in controlrats (FIG. 40). In fact, rats receiving the ORO polypeptide with alum orMN51 exhibited 90-100 percent reductions in the amount of free rat IgE(FIG. 41). These results demonstrate that chimeric IgE polypeptides incombination with alum or MN51 can reduce the levels of free IgE presentwithin a mammal.

Example 7

[0152] Studies in Cynomoigus Monkeys

[0153] The ability of the compositions provided herein to elicit ananti-self IgE antibody response also was examined in cynomolgus monkeys.H-OCO-H and H-OCOCO-H polypeptides were prepared that were similar tothe ORO and ORORO polypeptides described herein, with the exception thatthe rat IgE segments were replaced with IgE segments from cynomolgusmonkey. Groups of 5 or 6 animals were subcutaneously immunized withvehicle (PBS) plus MN51, 500 μg H-OCO-H plus MN51, or 500 μg H-OCOCO-Hplus MN51 (FIG. 42). Booster immunizations (300 μg) were given at weeks3 and 7, while re-boosters (300 μg) were given at weeks 29 and 32. Bloodsamples were obtained at weeks −1, 5, 9, 12, 15, 18, 21, 24, 27, 32 and35. The anti-IgE responses were measured against a recombinant part ofthe constant domain (Cε2-Cε3-Cε4) of cynomolgus monkey IgE (FIG. 43).Titer dilution₅₀ values were measured for each week that samples wereobtained.

[0154] Immunization with either H-OCO-H or H-OCOCO-H resulted in anincrease in titer dilution₅₀ that reached a maximum level at 9 or 10weeks and then decreased (FIG. 44). H-OCOCO-H induced a slightly greatereffect than H-OCO-H, although the difference was not significant (FIG.44). The study using the H-OCOCO-H group was terminated at week 18.

[0155] The anti-IgE response to H-OCO-H decreased over time,demonstrating that the effect is reversible (FIG. 44). To determinewhether the anti-IgE response is repeatable, the previously vaccinatedanimals were challenged with H-OCO-H at weeks 29 and 32. Animalspreviously exhibiting an anti-IgE response exhibited a second anti-IgEresponse (FIG. 44). The H-OCO-H and H-OCOCO-H vaccines did not produceunwanted haematological effects on thrombocyte counts (FIG. 45) or otherblood cells (FIG. 46). This experiment demonstrated that compositionscontaining a polypeptide such as H-OCO-H or H-OCOCO-H can be used incombination with MN51 to stimulate an anti-self IgE antibody response inprimates, and that similar compositions could be developed for use inhumans.

[0156] In another experiment, groups of cynomolgus monkeys weresubcutaneously immunized with H-OCO-H in combination with eitherAlhydrogel™ 1.3% (an aluminum hydroxide gel adjuvant; Brenntag StinnesLogistics) or MN51 (FIG. 47). Control monkeys were immunized with salinein combination with Alhydrogel™ 1.3%. Booster immunizations were givenat weeks 3, 5, and 7, and blood samples were obtained at weeks −1, 3, 5,7, 9, 12, 15, and 18. The amount of monkey anti-IgE antibodies measuredin monkeys receiving the H-OCO-H polypeptide were significantlyincreased as compared to the amounts measured in control monkeys (FIG.48). In addition, monkeys receiving the H-OCO-H polypeptide incombination with alum produced a stronger anti-IgE antibody responsethan the response produced by monkeys treated with the H-OCO-Hpolypeptide in combination with MN51 (FIG. 48).

[0157] These results demonstrate that chimeric IgE polypeptides withalum or MN51 can break a primate's self tolerance to IgE. These resultsalso demonstrate that the anti-IgE responses are reversible andrepeatable. In addition, primates treated with chimeric IgE polypeptideswith alum or MN51 exhibited no signs of thrombocytopenia or otherunwanted hematological effects.

Other Embodiments

[0158] It is to be understood that while the invention has beendescribed in conjunction with the detailed description thereof, theforegoing description is intended to illustrate and not limit the scopeof the invention, which is defined by the scope of the appended claims.Other aspects, advantages, and modifications are within the scope of thefollowing claims.

1 24 1 6649 DNA Artificial Sequence vector sequence 1 tcaatattggccattagcca tattattcat tggttatata gcataaatca atattggcta 60 ttggccattgcatacgttgt atctatatca taatatgtac atttatattg gctcatgtcc 120 aatatgaccgccatgttggc attgattatt gactagttat taatagtaat caattacggg 180 gtcattagttcatagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc 240 gcctggctgaccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat 300 agtaacgccaatagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc 360 ccacttggcagtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga 420 cggtaaatggcccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg 480 gcagtacatctacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac 540 caatgggcgtggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt 600 caatgggagtttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactg 660 cgatcgcccgccccgttgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata 720 agcagagctcgtttagtgaa ccgtcagatc actagaagct ttattgcggt agtttatcac 780 agttaaattgctaacgcagt cagtgcttct gacacaacag tctcgaactt aagctgcagt 840 gactctcttaaggtagcctt gcagaagttg gtcgtgaggc actgggcagg taagtatcaa 900 ggttacaagacaggtttaag gagaccaata gaaactgggc ttgtcgagac agagaagact 960 cttgcgtttctgataggcac ctattggtct tactgacatc cactttgcct ttctctccac 1020 aggtgtccactcccagttca attacagctc ttaaggctag agtacttaat acgactcact 1080 ataggctagcctcgagaatt cacgcgtggt acctctagag tcgaccccgg gccgacctca 1140 ccatgggatggagctgtatc atcctcttct tggtagcaac agctacaggt aaggggctca 1200 cagtagcaggcttgaggtct ggacatatat atgggtgaca atgacatcca ctttgccttt 1260 ctctccacaggtgtgcattc ctcgagtact ttatctctcc cagaaagtgg ccctgtgaca 1320 atcatcccacctacagtgaa gctcttccac tcatcctgtg acccccgagg ggatgctcat 1380 tccaccatccagctgctctg ccttgtctct ggcttctccc cagccaaggt ccatgtgacc 1440 tggctggtagatggacagga ggctgaaaat ctctttccct atacaaccag acctaagagg 1500 gaagggggacagactttttc tctacaaagt gaagtcaaca tcacacaggg ccagtggatg 1560 tcatcaaacacctacacctg ccatgtcaag cacaatggca gcatctttga agacagttct 1620 agaagatgctcagatgatga gccccggggt gtgattacct acctgatccc acccagtccc 1680 ctcgacctgtatgaaaatgg gactcccaaa cttacctgtc tggttttgga cctggaaagt 1740 gaggagaatatcaccgtgac gtgggtccga gagcgtaaga agtctatagg ttcggcatcc 1800 cagaggagtaccaagcacca taatgccaca accagtatca cctccatctt gccagtggat 1860 gccaaggactggatcgaagg tgaaggctac cagtgcagag tggaccaccc tcactttccc 1920 aagcccattgtgcgttccat caccaagctt gctagcccag gcaaacgctt agcccccgag 1980 gtatatatgctccctccatc tccagaggaa acaggaacca ctcgcactgt aacctgccta 2040 attcggggtttctacccttc tgaaatatct gtccaatggc tgtttaataa cgaagaggac 2100 cacactggacaccatactac cacccgtccc caaaaggacc acggaacgga tccttccttc 2160 ttcctctacagccgaatgct tgtcaacaag tctatttggg aaaaaggcaa tctcgtcacc 2220 tgccgtgtggtgcatgaagc cctacctggc tcccgcaccc tggaaaaaag cctgcattac 2280 tcagctggtaactaatctcg agcagggcgg ccgcttccct ttagtgaggg ttaatgcttc 2340 gagcagacatgataagatac attgatgagt ttggacaaac cacaactaga atgcagtgaa 2400 aaaaatgctttatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct 2460 gcaataaacaagttaacaac aacaattgca ttcattttat gtttcaggtt cagggggaga 2520 tgtgggaggttttttaaagc aagtaaaacc tctacaaatg tggtaaaatc cgataaggat 2580 cgatccgggctggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg 2640 cagcctgaatggcgaatgga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg 2700 gttacgcgcagcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc 2760 ttcccttcctttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc 2820 cctttagggttccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt 2880 gatggttcacgtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag 2940 tccacgttctttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg 3000 gtctattcttttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag 3060 ctgatttaacaaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttcctga 3120 tgcggtattttctccttacg catctgtgcg gtatttcaca ccgcatacgc ggatctgcgc 3180 agcaccatggcctgaaataa cctctgaaag aggaacttgg ttaggtacct tctgaggcgg 3240 aaagaaccagctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc 3300 aggcagaagtatgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc 3360 aggctccccagcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt 3420 cccgcccctaactccgccca tcccgcccct aactccgccc agttccgccc attctccgcc 3480 ccatggctgactaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct 3540 attccagaagtagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agcttgattc 3600 ttctgacacaacagtctcga acttaaggct agagccacca tgattgaaca agatggattg 3660 cacgcaggttctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag 3720 acaatcggctgctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt 3780 tttgtcaagaccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta 3840 tcgtggctggccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg 3900 ggaagggactggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt 3960 gctcctgccgagaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat 4020 ccggctacctgcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg 4080 atggaagccggtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca 4140 gccgaactgttcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc 4200 catggcgatgcctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc 4260 gactgtggccggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat 4320 attgctgaagagcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc 4380 gctcccgattcgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga 4440 ctctggggttcgaaatgacc gaccaagcga cgcccaacct gccatcacga tggccgcaat 4500 aaaatatctttattttcatt acatctgtgt gttggttttt tgtgtgaatc gatagcgata 4560 aggatccgcgtatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag 4620 ccccgacacccgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc 4680 gcttacagacaagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca 4740 tcaccgaaacgcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc 4800 atgataataatggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc 4860 cctatttgtttatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc 4920 tgataaatgcttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc 4980 gcccttattcccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg 5040 gtgaaagtaaaagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat 5100 ctcaacagcggtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc 5160 acttttaaagttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa 5220 ctcggtcgccgcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa 5280 aagcatcttacggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt 5340 gataacactgcggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct 5400 tttttgcacaacatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat 5460 gaagccataccaaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg 5520 cgcaaactattaactggcga actacttact ctagcttccc ggcaacaatt aatagactgg 5580 atggaggcggataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt 5640 attgctgataaatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg 5700 ccagatggtaagccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg 5760 gatgaacgaaatagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg 5820 tcagaccaagtttactcata tatactttag attgatttaa aacttcattt ttaatttaaa 5880 aggatctaggtgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt 5940 tcgttccactgagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt 6000 tttctgcgcgtaatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt 6060 ttgccggatcaagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag 6120 ataccaaatactgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta 6180 gcaccgcctacatacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat 6240 aagtcgtgtcttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg 6300 ggctgaacggggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg 6360 agatacctacagcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac 6420 aggtatccggtaagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga 6480 aacgcctggtatctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt 6540 ttgtgatgctcgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta 6600 cggttcctggccttttgctg gccttttgct cacatggctc gacagatct 6649 2 1011 DNA ArtificialSequence insert sequence 2 tcgagtactt tatctctccc agaaagtggc cctgtgacaatcatcccacc tacagtgaag 60 ctcttccact catcctgtga cccccgaggg gatgctcattccaccatcca gctgctctgc 120 cttgtctctg gcttctcccc agccaaggtc catgtgacctggctggtaga tggacaggag 180 gctgaaaatc tctttcccta tacaaccaga cctaagagggaagggggaca gactttttct 240 ctacaaagtg aagtcaacat cacacagggc cagtggatgtcatcaaacac ctacacctgc 300 catgtcaagc acaatggcag catctttgaa gacagttctagaagatgctc agatgatgag 360 ccccggggtg tgattaccta cctgatccca cccagtcccctcgacctgta tgaaaatggg 420 actcccaaac ttacctgtct ggttttggac ctggaaagtgaggagaatat caccgtgacg 480 tgggtccgag agcgtaagaa gtctataggt tcggcatcccagaggagtac caagcaccat 540 aatgccacaa ccagtatcac ctccatcttg ccagtggatgccaaggactg gatcgaaggt 600 gaaggctacc agtgcagagt ggaccaccct cactttcccaagcccattgt gcgttccatc 660 accaagcttg ctagcccagg caaacgctta gcccccgaggtatatatgct ccctccatct 720 ccagaggaaa caggaaccac tcgcactgta acctgcctaattcggggttt ctacccttct 780 gaaatatctg tccaatggct gtttaataac gaagaggaccacactggaca ccatactacc 840 acccgtcccc aaaaggacca cggaacggat ccttccttcttcctctacag ccgaatgctt 900 gtcaacaagt ctatttggga aaaaggcaat ctcgtcacctgccgtgtggt gcatgaagcc 960 ctacctggct cccgcaccct ggaaaaaagc ctgcattactcagctggtaa c 1011 3 337 PRT Artificial Sequence chimeric polypeptide 3Ser Ser Thr Leu Ser Leu Pro Glu Ser Gly Pro Val Thr Ile Ile Pro 1 5 1015 Pro Thr Val Lys Leu Phe His Ser Ser Cys Asp Pro Arg Gly Asp Ala 20 2530 His Ser Thr Ile Gln Leu Leu Cys Leu Val Ser Gly Phe Ser Pro Ala 35 4045 Lys Val His Val Thr Trp Leu Val Asp Gly Gln Glu Ala Glu Asn Leu 50 5560 Phe Pro Tyr Thr Thr Arg Pro Lys Arg Glu Gly Gly Gln Thr Phe Ser 65 7075 80 Leu Gln Ser Glu Val Asn Ile Thr Gln Gly Gln Trp Met Ser Ser Asn 8590 95 Thr Tyr Thr Cys His Val Lys His Asn Gly Ser Ile Phe Glu Asp Ser100 105 110 Ser Arg Arg Cys Ser Asp Asp Glu Pro Arg Gly Val Ile Thr TyrLeu 115 120 125 Ile Pro Pro Ser Pro Leu Asp Leu Tyr Glu Asn Gly Thr ProLys Leu 130 135 140 Thr Cys Leu Val Leu Asp Leu Glu Ser Glu Glu Asn IleThr Val Thr 145 150 155 160 Trp Val Arg Glu Arg Lys Lys Ser Ile Gly SerAla Ser Gln Arg Ser 165 170 175 Thr Lys His His Asn Ala Thr Thr Ser IleThr Ser Ile Leu Pro Val 180 185 190 Asp Ala Lys Asp Trp Ile Glu Gly GluGly Tyr Gln Cys Arg Val Asp 195 200 205 His Pro His Phe Pro Lys Pro IleVal Arg Ser Ile Thr Lys Leu Ala 210 215 220 Ser Pro Gly Lys Arg Leu AlaPro Glu Val Tyr Met Leu Pro Pro Ser 225 230 235 240 Pro Glu Glu Thr GlyThr Thr Arg Thr Val Thr Cys Leu Ile Arg Gly 245 250 255 Phe Tyr Pro SerGlu Ile Ser Val Gln Trp Leu Phe Asn Asn Glu Glu 260 265 270 Asp His ThrGly His His Thr Thr Thr Arg Pro Gln Lys Asp His Gly 275 280 285 Thr AspPro Ser Phe Phe Leu Tyr Ser Arg Met Leu Val Asn Lys Ser 290 295 300 IleTrp Glu Lys Gly Asn Leu Val Thr Cys Arg Val Val His Glu Ala 305 310 315320 Leu Pro Gly Ser Arg Thr Leu Glu Lys Ser Leu His Tyr Ser Ala Gly 325330 335 Asn 4 6652 DNA Artificial Sequence vector sequence 4 tcaatattggccattagcca tattattcat tggttatata gcataaatca atattggcta 60 ttggccattgcatacgttgt atctatatca taatatgtac atttatattg gctcatgtcc 120 aatatgaccgccatgttggc attgattatt gactagttat taatagtaat caattacggg 180 gtcattagttcatagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc 240 gcctggctgaccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat 300 agtaacgccaatagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc 360 ccacttggcagtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga 420 cggtaaatggcccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg 480 gcagtacatctacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac 540 caatgggcgtggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt 600 caatgggagtttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactg 660 cgatcgcccgccccgttgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata 720 agcagagctcgtttagtgaa ccgtcagatc actagaagct ttattgcggt agtttatcac 780 agttaaattgctaacgcagt cagtgcttct gacacaacag tctcgaactt aagctgcagt 840 gactctcttaaggtagcctt gcagaagttg gtcgtgaggc actgggcagg taagtatcaa 900 ggttacaagacaggtttaag gagaccaata gaaactgggc ttgtcgagac agagaagact 960 cttgcgtttctgataggcac ctattggtct tactgacatc cactttgcct ttctctccac 1020 aggtgtccactcccagttca attacagctc ttaaggctag agtacttaat acgactcact 1080 ataggctagcctcgagaatt cacgcgtggt acctctagag tcgaccccgg gccgacctca 1140 ccatgggatggagctgtatc atcctcttct tggtagcaac agctacaggt aaggggctca 1200 cagtagcaggcttgaggtct ggacatatat atgggtgaca atgacatcca ctttgccttt 1260 ctctccacaggtgtgcattc ctcgagtact ttatctctcc cagaaagtgg ccctgtgaca 1320 atcatcccacctacagtgaa gctcttccac tcatcctgtg acccccgagg ggatgctcat 1380 tccaccatccagctgctctg ccttgtctct ggcttctccc cagccaaggt ccatgtgacc 1440 tggctggtagatggacagga ggctgaaaat ctctttccct atacaaccag acctaagagg 1500 gaagggggacagactttttc tctacaaagt gaagtcaaca tcacacaggg ccagtggatg 1560 tcatcaaacacctacacctg ccatgtcaag cacaatggca gcatctttga agacagttct 1620 agaaagtgtgcagattccaa cccgagaggg gtgagcgcct acctaagccg gcccagcccg 1680 ttcgacctgttcatccgcaa gtcgcccacg atcacctgtc tggtggtgga cctggcaccc 1740 agcaaggggaccgtgaacct gacctggtcc cgggccagtg ggaagcctgt gaaccactcc 1800 accagaaaggaggagaagca gcgcaatggc acgttaaccg tcacgtccac cctgccggtg 1860 ggcacccgagactggatcga gggggagacc taccagtgca gggtgaccca cccccacctg 1920 cccagggccctcatgcggtc cacgaccaag cttgctagcc caggcaaacg cttagccccc 1980 gaggtatatatgctccctcc atctccagag gaaacaggaa ccactcgcac tgtaacctgc 2040 ctaattcggggtttctaccc ttctgaaata tctgtccaat ggctgtttaa taacgaagag 2100 gaccacactggacaccatac taccacccgt ccccaaaagg accacggaac ggatccttcc 2160 ttcttcctctacagccgaat gcttgtcaac aagtctattt gggaaaaagg caatctcgtc 2220 acctgccgtgtggtgcatga agccctacct ggctcccgca ccctggaaaa aagcctgcat 2280 tactcagctggtaactaatc tcgagcaggg cggccgcttc cctttagtga gggttaatgc 2340 ttcgagcagacatgataaga tacattgatg agtttggaca aaccacaact agaatgcagt 2400 gaaaaaaatgctttatttgt gaaatttgtg atgctattgc tttatttgta accattataa 2460 gctgcaataaacaagttaac aacaacaatt gcattcattt tatgtttcag gttcaggggg 2520 agatgtgggaggttttttaa agcaagtaaa acctctacaa atgtggtaaa atccgataag 2580 gatcgatccgggctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt 2640 gcgcagcctgaatggcgaat ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg 2700 gtggttacgcgcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct 2760 ttcttcccttcctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg 2820 ctccctttagggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag 2880 ggtgatggttcacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg 2940 gagtccacgttctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc 3000 tcggtctattcttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat 3060 gagctgatttaacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttcc 3120 tgatgcggtattttctcctt acgcatctgt gcggtatttc acaccgcata cgcggatctg 3180 cgcagcaccatggcctgaaa taacctctga aagaggaact tggttaggta ccttctgagg 3240 cggaaagaaccagctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc 3300 agcaggcagaagtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc 3360 cccaggctccccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat 3420 agtcccgcccctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc 3480 gccccatggctgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga 3540 gctattccagaagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagcttga 3600 ttcttctgacacaacagtct cgaacttaag gctagagcca ccatgattga acaagatgga 3660 ttgcacgcaggttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa 3720 cagacaatcggctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt 3780 ctttttgtcaagaccgacct gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg 3840 ctatcgtggctggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa 3900 gcgggaagggactggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac 3960 cttgctcctgccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt 4020 gatccggctacctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact 4080 cggatggaagccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg 4140 ccagccgaactgttcgccag gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg 4200 acccatggcgatgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc 4260 atcgactgtggccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt 4320 gatattgctgaagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc 4380 gccgctcccgattcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgagcg 4440 ggactctggggttcgaaatg accgaccaag cgacgcccaa cctgccatca cgatggccgc 4500 aataaaatatctttattttc attacatctg tgtgttggtt ttttgtgtga atcgatagcg 4560 ataaggatccgcgtatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc 4620 cagccccgacacccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca 4680 tccgcttacagacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg 4740 tcatcaccgaaacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat 4800 gtcatgataataatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga 4860 acccctatttgtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa 4920 ccctgataaatgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt 4980 gtcgcccttattcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg 5040 ctggtgaaagtaaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg 5100 gatctcaacagcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg 5160 agcacttttaaagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag 5220 caactcggtcgccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca 5280 gaaaagcatcttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg 5340 agtgataacactgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc 5400 gcttttttgcacaacatggg ggatcatgta actcgccttg atcgttggga accggagctg 5460 aatgaagccataccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg 5520 ttgcgcaaactattaactgg cgaactactt actctagctt cccggcaaca attaatagac 5580 tggatggaggcggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg 5640 tttattgctgataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg 5700 gggccagatggtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact 5760 atggatgaacgaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa 5820 ctgtcagaccaagtttactc atatatactt tagattgatt taaaacttca tttttaattt 5880 aaaaggatctaggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag 5940 ttttcgttccactgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct 6000 ttttttctgcgcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt 6060 tgtttgccggatcaagagct accaactctt tttccgaagg taactggctt cagcagagcg 6120 cagataccaaatactgttct tctagtgtag ccgtagttag gccaccactt caagaactct 6180 gtagcaccgcctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc 6240 gataagtcgtgtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg 6300 tcgggctgaacggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa 6360 ctgagatacctacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg 6420 gacaggtatccggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg 6480 ggaaacgcctggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga 6540 tttttgtgatgctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt 6600 ttacggttcctggccttttg ctggcctttt gctcacatgg ctcgacagat ct 6652 5 1014 DNAArtificial Sequence insert sequence 5 tcgagtactt tatctctccc agaaagtggccctgtgacaa tcatcccacc tacagtgaag 60 ctcttccact catcctgtga cccccgaggggatgctcatt ccaccatcca gctgctctgc 120 cttgtctctg gcttctcccc agccaaggtccatgtgacct ggctggtaga tggacaggag 180 gctgaaaatc tctttcccta tacaaccagacctaagaggg aagggggaca gactttttct 240 ctacaaagtg aagtcaacat cacacagggccagtggatgt catcaaacac ctacacctgc 300 catgtcaagc acaatggcag catctttgaagacagttcta gaaagtgtgc agattccaac 360 ccgagagggg tgagcgccta cctaagccggcccagcccgt tcgacctgtt catccgcaag 420 tcgcccacga tcacctgtct ggtggtggacctggcaccca gcaaggggac cgtgaacctg 480 acctggtccc gggccagtgg gaagcctgtgaaccactcca ccagaaagga ggagaagcag 540 cgcaatggca cgttaaccgt cacgtccaccctgccggtgg gcacccgaga ctggatcgag 600 ggggagacct accagtgcag ggtgacccacccccacctgc ccagggccct catgcggtcc 660 acgaccaagc ttgctagccc aggcaaacgcttagcccccg aggtatatat gctccctcca 720 tctccagagg aaacaggaac cactcgcactgtaacctgcc taattcgggg tttctaccct 780 tctgaaatat ctgtccaatg gctgtttaataacgaagagg accacactgg acaccatact 840 accacccgtc cccaaaagga ccacggaacggatccttcct tcttcctcta cagccgaatg 900 cttgtcaaca agtctatttg ggaaaaaggcaatctcgtca cctgccgtgt ggtgcatgaa 960 gccctacctg gctcccgcac cctggaaaaaagcctgcatt actcagctgg taac 1014 6 338 PRT Artificial Sequence chimericpolypeptide 6 Ser Ser Thr Leu Ser Leu Pro Glu Ser Gly Pro Val Thr IleIle Pro 1 5 10 15 Pro Thr Val Lys Leu Phe His Ser Ser Cys Asp Pro ArgGly Asp Ala 20 25 30 His Ser Thr Ile Gln Leu Leu Cys Leu Val Ser Gly PheSer Pro Ala 35 40 45 Lys Val His Val Thr Trp Leu Val Asp Gly Gln Glu AlaGlu Asn Leu 50 55 60 Phe Pro Tyr Thr Thr Arg Pro Lys Arg Glu Gly Gly GlnThr Phe Ser 65 70 75 80 Leu Gln Ser Glu Val Asn Ile Thr Gln Gly Gln TrpMet Ser Ser Asn 85 90 95 Thr Tyr Thr Cys His Val Lys His Asn Gly Ser IlePhe Glu Asp Ser 100 105 110 Ser Arg Lys Cys Ala Asp Ser Asn Pro Arg GlyVal Ser Ala Tyr Leu 115 120 125 Ser Arg Pro Ser Pro Phe Asp Leu Phe IleArg Lys Ser Pro Thr Ile 130 135 140 Thr Cys Leu Val Val Asp Leu Ala ProSer Lys Gly Thr Val Asn Leu 145 150 155 160 Thr Trp Ser Arg Ala Ser GlyLys Pro Val Asn His Ser Thr Arg Lys 165 170 175 Glu Glu Lys Gln Arg AsnGly Thr Leu Thr Val Thr Ser Thr Leu Pro 180 185 190 Val Gly Thr Arg AspTrp Ile Glu Gly Glu Thr Tyr Gln Cys Arg Val 195 200 205 Thr His Pro HisLeu Pro Arg Ala Leu Met Arg Ser Thr Thr Lys Leu 210 215 220 Ala Ser ProGly Lys Arg Leu Ala Pro Glu Val Tyr Met Leu Pro Pro 225 230 235 240 SerPro Glu Glu Thr Gly Thr Thr Arg Thr Val Thr Cys Leu Ile Arg 245 250 255Gly Phe Tyr Pro Ser Glu Ile Ser Val Gln Trp Leu Phe Asn Asn Glu 260 265270 Glu Asp His Thr Gly His His Thr Thr Thr Arg Pro Gln Lys Asp His 275280 285 Gly Thr Asp Pro Ser Phe Phe Leu Tyr Ser Arg Met Leu Val Asn Lys290 295 300 Ser Ile Trp Glu Lys Gly Asn Leu Val Thr Cys Arg Val Val HisGlu 305 310 315 320 Ala Leu Pro Gly Ser Arg Thr Leu Glu Lys Ser Leu HisTyr Ser Ala 325 330 335 Gly Asn 7 1665 DNA Artificial Sequence insertsequence 7 tcgagtactt tatctctccc agaaagtggc cctgtgacaa tcatcccacctacagtgaag 60 ctcttccact catcctgtga cccccgaggg gatgctcatt ccaccatccagctgctctgc 120 cttgtctctg gcttctcccc agccaaggtc catgtgacct ggctggtagatggacaggag 180 gctgaaaatc tctttcccta tacaaccaga cctaagaggg aagggggacagactttttct 240 ctacaaagtg aagtcaacat cacacagggc cagtggatgt catcaaacacctacacctgc 300 catgtcaagc acaatggcag catctttgaa gacagttcta gaagatgctcagatgatgag 360 ccccggggtg tgattaccta cctgatccca cccagtcccc tcgacctgtatgaaaatggg 420 actcccaaac ttacctgtct ggttttggac ctggaaagtg aggagaatatcaccgtgacg 480 tgggtccgag agcgtaagaa gtctataggt tcggcatccc agaggagtaccaagcaccat 540 aatgccacaa ccagtatcac ctccatcttg ccagtggatg ccaaggactggatcgaaggt 600 gaaggctacc agtgcagagt ggaccaccct cactttccca agcccattgtgcgttccatc 660 accaagctta tcgatctccc agaaagtggc cctgtgacaa tcatcccacctacagtgaag 720 ctcttccact catcctgtga cccccgaggg gatgctcatt ccaccatccagctgctctgc 780 cttgtctctg gcttctcccc agccaaggtc catgtgacct ggctggtagatggacaggag 840 gctgaaaatc tctttcccta tacaaccaga cctaagaggg aagggggacagactttttct 900 ctacaaagtg aagtcaacat cacacagggc cagtggatgt catcaaacacctacacctgc 960 catgtcaagc acaatggcag catctttgaa gacagttcta gaagatgctcagatgatgag 1020 ccccggggtg tgattaccta cctgatccca cccagtcccc tcgacctgtatgaaaatggg 1080 actcccaaac ttacctgtct ggttttggac ctggaaagtg aggagaatatcaccgtgacg 1140 tgggtccgag agcgtaagaa gtctataggt tcggcatccc agaggagtaccaagcaccat 1200 aatgccacaa ccagtatcac ctccatcttg ccagtggatg ccaaggactggatcgaaggt 1260 gaaggctacc agtgcagagt ggaccaccct cactttccca agcccattgtgcgttccatc 1320 accgctagcc caggcaaacg cttagccccc gaggtatata tgctccctccatctccagag 1380 gaaacaggaa ccactcgcac tgtaacctgc ctaattcggg gtttctacccttctgaaata 1440 tctgtccaat ggctgtttaa taacgaagag gaccacactg gacaccatactaccacccgt 1500 ccccaaaagg accacggaac ggatccttcc ttcttcctct acagccgaatgcttgtcaac 1560 aagtctattt gggaaaaagg caatctcgtc acctgccgtg tggtgcatgaagccctacct 1620 ggctcccgca ccctggaaaa aagcctgcat tactcagctg gtaac 1665 8555 PRT Artificial Sequence chimeric polypeptide 8 Ser Ser Thr Leu SerLeu Pro Glu Ser Gly Pro Val Thr Ile Ile Pro 1 5 10 15 Pro Thr Val LysLeu Phe His Ser Ser Cys Asp Pro Arg Gly Asp Ala 20 25 30 His Ser Thr IleGln Leu Leu Cys Leu Val Ser Gly Phe Ser Pro Ala 35 40 45 Lys Val His ValThr Trp Leu Val Asp Gly Gln Glu Ala Glu Asn Leu 50 55 60 Phe Pro Tyr ThrThr Arg Pro Lys Arg Glu Gly Gly Gln Thr Phe Ser 65 70 75 80 Leu Gln SerGlu Val Asn Ile Thr Gln Gly Gln Trp Met Ser Ser Asn 85 90 95 Thr Tyr ThrCys His Val Lys His Asn Gly Ser Ile Phe Glu Asp Ser 100 105 110 Ser ArgArg Cys Ser Asp Asp Glu Pro Arg Gly Val Ile Thr Tyr Leu 115 120 125 IlePro Pro Ser Pro Leu Asp Leu Tyr Glu Asn Gly Thr Pro Lys Leu 130 135 140Thr Cys Leu Val Leu Asp Leu Glu Ser Glu Glu Asn Ile Thr Val Thr 145 150155 160 Trp Val Arg Glu Arg Lys Lys Ser Ile Gly Ser Ala Ser Gln Arg Ser165 170 175 Thr Lys His His Asn Ala Thr Thr Ser Ile Thr Ser Ile Leu ProVal 180 185 190 Asp Ala Lys Asp Trp Ile Glu Gly Glu Gly Tyr Gln Cys ArgVal Asp 195 200 205 His Pro His Phe Pro Lys Pro Ile Val Arg Ser Ile ThrLys Leu Ile 210 215 220 Asp Leu Pro Glu Ser Gly Pro Val Thr Ile Ile ProPro Thr Val Lys 225 230 235 240 Leu Phe His Ser Ser Cys Asp Pro Arg GlyAsp Ala His Ser Thr Ile 245 250 255 Gln Leu Leu Cys Leu Val Ser Gly PheSer Pro Ala Lys Val His Val 260 265 270 Thr Trp Leu Val Asp Gly Gln GluAla Glu Asn Leu Phe Pro Tyr Thr 275 280 285 Thr Arg Pro Lys Arg Glu GlyGly Gln Thr Phe Ser Leu Gln Ser Glu 290 295 300 Val Asn Ile Thr Gln GlyGln Trp Met Ser Ser Asn Thr Tyr Thr Cys 305 310 315 320 His Val Lys HisAsn Gly Ser Ile Phe Glu Asp Ser Ser Arg Arg Cys 325 330 335 Ser Asp AspGlu Pro Arg Gly Val Ile Thr Tyr Leu Ile Pro Pro Ser 340 345 350 Pro LeuAsp Leu Tyr Glu Asn Gly Thr Pro Lys Leu Thr Cys Leu Val 355 360 365 LeuAsp Leu Glu Ser Glu Glu Asn Ile Thr Val Thr Trp Val Arg Glu 370 375 380Arg Lys Lys Ser Ile Gly Ser Ala Ser Gln Arg Ser Thr Lys His His 385 390395 400 Asn Ala Thr Thr Ser Ile Thr Ser Ile Leu Pro Val Asp Ala Lys Asp405 410 415 Trp Ile Glu Gly Glu Gly Tyr Gln Cys Arg Val Asp His Pro HisPhe 420 425 430 Pro Lys Pro Ile Val Arg Ser Ile Thr Ala Ser Pro Gly LysArg Leu 435 440 445 Ala Pro Glu Val Tyr Met Leu Pro Pro Ser Pro Glu GluThr Gly Thr 450 455 460 Thr Arg Thr Val Thr Cys Leu Ile Arg Gly Phe TyrPro Ser Glu Ile 465 470 475 480 Ser Val Gln Trp Leu Phe Asn Asn Glu GluAsp His Thr Gly His His 485 490 495 Thr Thr Thr Arg Pro Gln Lys Asp HisGly Thr Asp Pro Ser Phe Phe 500 505 510 Leu Tyr Ser Arg Met Leu Val AsnLys Ser Ile Trp Glu Lys Gly Asn 515 520 525 Leu Val Thr Cys Arg Val ValHis Glu Ala Leu Pro Gly Ser Arg Thr 530 535 540 Leu Glu Lys Ser Leu HisTyr Ser Ala Gly Asn 545 550 555 9 1698 DNA Artificial Sequence insertsequence 9 tcgagtactt tatctctccc agaaagtggc cctgtgacaa tcatcccacctacagtgaag 60 ctcttccact catcctgtga cccccgaggg gatgctcatt ccaccatccagctgctctgc 120 cttgtctctg gcttctcccc agccaaggtc catgtgacct ggctggtagatggacaggag 180 gctgaaaatc tctttcccta tacaaccaga cctaagaggg aagggggacagactttttct 240 ctacaaagtg aagtcaacat cacacagggc cagtggatgt catcaaacacctacacctgc 300 catgtcaagc acaatggcag catctttgaa gacagttcta gaaagtgtgcagattccaac 360 ccgagagggg tgagcgccta cctaagccgg cccagcccgt tcgacctgttcatccgcaag 420 tcgcccacga tcacctgtct ggtggtggac ctggcaccca gcaaggggaccgtgaacctg 480 acctggtccg aggcccaagg gaagcctgtg aaccactcca ccagaaaggaggagaagcag 540 cgcaatggca cgttaaccgt cacgtccacc ctgccggtgg gcacccgagactggatcgag 600 gggcgtacgt accagtgcag ggtgacccac ccccacctgc ccagggccctcatgcggtcc 660 acgaccaagc ttatcgatat cccagaaagt ggccctgtga caatcatcccacctacagtg 720 aagctcttcc actcatcctg tgacccccga ggggatgctc attccaccatccagctgctc 780 tgccttgtct ctggcttctc cccagccaag gtccatgtga cctggctggtagatggacag 840 gaggctgaaa atctctttcc ctatacaacc agacctaaga gggaagggggacagactttt 900 tctctacaaa gtgaagtcaa catcacacag ggccagtgga tgtcatcaaacacctacacc 960 tgccatgtca agcacaatgg cagcatcttt gaagacagtt ctagaaagtgtgcagattcc 1020 aacccgagag gggtgagcgc ctacctaagc cggcccagcc cgttcgacctgttcatccgc 1080 aagtcgccca cgatcacctg tctggtggtg gacctggcac ccagcaaggggaccgtgaac 1140 ctgacctggt ccgaggccca agggaagcct gtgaaccact ccaccagaaaggaggagaag 1200 cagcgcaatg gcacgttaac cgtcacgtcc accctgccgg tgggcacccgagactggatc 1260 gaggggcgta cgtaccagtg cagggtgacc cacccccacc tgcccagggccctcatgcgg 1320 tccacgaccg ctagcccagg caaacgctta gcccccgagg tatatatgctccctccatct 1380 ccagaggaaa caggaaccac tcgcactgta acctgcctaa ttcggggtttctacccttct 1440 gaaatatctg tccaatggct gtttaataac gaagaggacc acactggacaccatactacc 1500 acccgtcccc aaaaggacca cggaacggat ccttccttct tcctctacagccgaatgctt 1560 gtcaacaagt ctatttggga aaaaggcaat ctcgtcacct gccgtgtggtgcatgaagcc 1620 ctacctggct cccgcaccct ggaaaaaagc ctgcattact cagctggtaacggatcagga 1680 caccatcacc atcaccat 1698 10 566 PRT Artificial Sequencechimeric polypeptide 10 Ser Ser Thr Leu Ser Leu Pro Glu Ser Gly Pro ValThr Ile Ile Pro 1 5 10 15 Pro Thr Val Lys Leu Phe His Ser Ser Cys AspPro Arg Gly Asp Ala 20 25 30 His Ser Thr Ile Gln Leu Leu Cys Leu Val SerGly Phe Ser Pro Ala 35 40 45 Lys Val His Val Thr Trp Leu Val Asp Gly GlnGlu Ala Glu Asn Leu 50 55 60 Phe Pro Tyr Thr Thr Arg Pro Lys Arg Glu GlyGly Gln Thr Phe Ser 65 70 75 80 Leu Gln Ser Glu Val Asn Ile Thr Gln GlyGln Trp Met Ser Ser Asn 85 90 95 Thr Tyr Thr Cys His Val Lys His Asn GlySer Ile Phe Glu Asp Ser 100 105 110 Ser Arg Lys Cys Ala Asp Ser Asn ProArg Gly Val Ser Ala Tyr Leu 115 120 125 Ser Arg Pro Ser Pro Phe Asp LeuPhe Ile Arg Lys Ser Pro Thr Ile 130 135 140 Thr Cys Leu Val Val Asp LeuAla Pro Ser Lys Gly Thr Val Asn Leu 145 150 155 160 Thr Trp Ser Arg AlaSer Gly Lys Pro Val Asn His Ser Thr Arg Lys 165 170 175 Glu Glu Lys GlnArg Asn Gly Thr Leu Thr Val Thr Ser Thr Leu Pro 180 185 190 Val Gly ThrArg Asp Trp Ile Glu Gly Glu Thr Tyr Gln Cys Arg Val 195 200 205 Thr HisPro His Leu Pro Arg Ala Leu Met Arg Ser Thr Thr Lys Leu 210 215 220 IleAsp Ile Pro Glu Ser Gly Pro Val Thr Ile Ile Pro Pro Thr Val 225 230 235240 Lys Leu Phe His Ser Ser Cys Asp Pro Arg Gly Asp Ala His Ser Thr 245250 255 Ile Gln Leu Leu Cys Leu Val Ser Gly Phe Ser Pro Ala Lys Val His260 265 270 Val Thr Trp Leu Val Asp Gly Gln Glu Ala Glu Asn Leu Phe ProTyr 275 280 285 Thr Thr Arg Pro Lys Arg Glu Gly Gly Gln Thr Phe Ser LeuGln Ser 290 295 300 Glu Val Asn Ile Thr Gln Gly Gln Trp Met Ser Ser AsnThr Tyr Thr 305 310 315 320 Cys His Val Lys His Asn Gly Ser Ile Phe GluAsp Ser Ser Arg Lys 325 330 335 Cys Ala Asp Ser Asn Pro Arg Gly Val SerAla Tyr Leu Ser Arg Pro 340 345 350 Ser Pro Phe Asp Leu Phe Ile Arg LysSer Pro Thr Ile Thr Cys Leu 355 360 365 Val Val Asp Leu Ala Pro Ser LysGly Thr Val Asn Leu Thr Trp Ser 370 375 380 Arg Ala Ser Gly Lys Pro ValAsn His Ser Thr Arg Lys Glu Glu Lys 385 390 395 400 Gln Arg Asn Gly ThrLeu Thr Val Thr Ser Thr Leu Pro Val Gly Thr 405 410 415 Arg Asp Trp IleGlu Gly Glu Thr Tyr Gln Cys Arg Val Thr His Pro 420 425 430 His Leu ProArg Ala Leu Met Arg Ser Thr Thr Ala Ser Pro Gly Lys 435 440 445 Arg LeuAla Pro Glu Val Tyr Met Leu Pro Pro Ser Pro Glu Glu Thr 450 455 460 GlyThr Thr Arg Thr Val Thr Cys Leu Ile Arg Gly Phe Tyr Pro Ser 465 470 475480 Glu Ile Ser Val Gln Trp Leu Phe Asn Asn Glu Glu Asp His Thr Gly 485490 495 His His Thr Thr Thr Arg Pro Gln Lys Asp His Gly Thr Asp Pro Ser500 505 510 Phe Phe Leu Tyr Ser Arg Met Leu Val Asn Lys Ser Ile Trp GluLys 515 520 525 Gly Asn Leu Val Thr Cys Arg Val Val His Glu Ala Leu ProGly Ser 530 535 540 Arg Thr Leu Glu Lys Ser Leu His Tyr Ser Ala Gly AsnGly Ser Gly 545 550 555 560 His His His His His His 565 11 1671 DNAArtificial Sequence insert sequence 11 tcgagtactt tatctctccc agaaagtggccctgtgacaa tcatcccacc tacagtgaag 60 ctcttccact catcctgtga cccccgaggggatgctcatt ccaccatcca gctgctctgc 120 cttgtctctg gcttctcccc agccaaggtccatgtgacct ggctggtaga tggacaggag 180 gctgaaaatc tctttcccta tacaaccagacctaagaggg aagggggaca gactttttct 240 ctacaaagtg aagtcaacat cacacagggccagtggatgt catcaaacac ctacacctgc 300 catgtcaagc acaatggcag catctttgaagacagttcta gaaagtgtgc agattccaac 360 ccgagagggg tgagcgccta cctaagccggcccagcccgt tcgacctgtt catccgcaag 420 tcgcccacga tcacctgtct ggtggtggacctggcaccca gcaaggggac cgtgaacctg 480 acctggtccg aggcccaagg gaagcctgtgaaccactcca ccagaaagga ggagaagcag 540 cgcaatggca cgttaaccgt cacgtccaccctgccggtgg gcacccgaga ctggatcgag 600 gggcgtacgt accagtgcag ggtgacccacccccacctgc ccagggccct catgcggtcc 660 acgaccaagc ttatcgatat cccagaaagtggccctgtga caatcatccc acctacagtg 720 aagctcttcc actcatcctg tgacccccgaggggatgctc attccaccat ccagctgctc 780 tgccttgtct ctggcttctc cccagccaaggtccatgtga cctggctggt agatggacag 840 gaggctgaaa atctctttcc ctatacaaccagacctaaga gggaaggggg acagactttt 900 tctctacaaa gtgaagtcaa catcacacagggccagtgga tgtcatcaaa cacctacacc 960 tgccatgtca agcacaatgg cagcatctttgaagacagtt ctagaaagtg tgcagattcc 1020 aacccgagag gggtgagcgc ctacctaagccggcccagcc cgttcgacct gttcatccgc 1080 aagtcgccca cgatcacctg tctggtggtggacctggcac ccagcaaggg gaccgtgaac 1140 ctgacctggt ccgaggccca agggaagcctgtgaaccact ccaccagaaa ggaggagaag 1200 cagcgcaatg gcacgttaac cgtcacgtccaccctgccgg tgggcacccg agactggatc 1260 gaggggcgta cgtaccagtg cagggtgacccacccccacc tgcccagggc cctcatgcgg 1320 tccacgaccg ctagcccagg caaacgcttagcccccgagg tatatatgct ccctccatct 1380 ccagaggaaa caggaaccac tcgcactgtaacctgcctaa ttcggggttt ctacccttct 1440 gaaatatctg tccaatggct gtttaataacgaagaggacc acactggaca ccatactacc 1500 acccgtcccc aaaaggacca cggaacggatccttccttct tcctctacag ccgaatgctt 1560 gtcaacaagt ctatttggga aaaaggcaatctcgtcacct gccgtgtggt gcatgaagcc 1620 ctacctggct cccgcaccct ggaaaaaagcctgcattact cagctggtaa c 1671 12 557 PRT Artificial Sequence chimericpolypeptide 12 Ser Ser Thr Leu Ser Leu Pro Glu Ser Gly Pro Val Thr IleIle Pro 1 5 10 15 Pro Thr Val Lys Leu Phe His Ser Ser Cys Asp Pro ArgGly Asp Ala 20 25 30 His Ser Thr Ile Gln Leu Leu Cys Leu Val Ser Gly PheSer Pro Ala 35 40 45 Lys Val His Val Thr Trp Leu Val Asp Gly Gln Glu AlaGlu Asn Leu 50 55 60 Phe Pro Tyr Thr Thr Arg Pro Lys Arg Glu Gly Gly GlnThr Phe Ser 65 70 75 80 Leu Gln Ser Glu Val Asn Ile Thr Gln Gly Gln TrpMet Ser Ser Asn 85 90 95 Thr Tyr Thr Cys His Val Lys His Asn Gly Ser IlePhe Glu Asp Ser 100 105 110 Ser Arg Lys Cys Ala Asp Ser Asn Pro Arg GlyVal Ser Ala Tyr Leu 115 120 125 Ser Arg Pro Ser Pro Phe Asp Leu Phe IleArg Lys Ser Pro Thr Ile 130 135 140 Thr Cys Leu Val Val Asp Leu Ala ProSer Lys Gly Thr Val Asn Leu 145 150 155 160 Thr Trp Ser Arg Ala Ser GlyLys Pro Val Asn His Ser Thr Arg Lys 165 170 175 Glu Glu Lys Gln Arg AsnGly Thr Leu Thr Val Thr Ser Thr Leu Pro 180 185 190 Val Gly Thr Arg AspTrp Ile Glu Gly Glu Thr Tyr Gln Cys Arg Val 195 200 205 Thr His Pro HisLeu Pro Arg Ala Leu Met Arg Ser Thr Thr Lys Leu 210 215 220 Ile Asp IlePro Glu Ser Gly Pro Val Thr Ile Ile Pro Pro Thr Val 225 230 235 240 LysLeu Phe His Ser Ser Cys Asp Pro Arg Gly Asp Ala His Ser Thr 245 250 255Ile Gln Leu Leu Cys Leu Val Ser Gly Phe Ser Pro Ala Lys Val His 260 265270 Val Thr Trp Leu Val Asp Gly Gln Glu Ala Glu Asn Leu Phe Pro Tyr 275280 285 Thr Thr Arg Pro Lys Arg Glu Gly Gly Gln Thr Phe Ser Leu Gln Ser290 295 300 Glu Val Asn Ile Thr Gln Gly Gln Trp Met Ser Ser Asn Thr TyrThr 305 310 315 320 Cys His Val Lys His Asn Gly Ser Ile Phe Glu Asp SerSer Arg Lys 325 330 335 Cys Ala Asp Ser Asn Pro Arg Gly Val Ser Ala TyrLeu Ser Arg Pro 340 345 350 Ser Pro Phe Asp Leu Phe Ile Arg Lys Ser ProThr Ile Thr Cys Leu 355 360 365 Val Val Asp Leu Ala Pro Ser Lys Gly ThrVal Asn Leu Thr Trp Ser 370 375 380 Arg Ala Ser Gly Lys Pro Val Asn HisSer Thr Arg Lys Glu Glu Lys 385 390 395 400 Gln Arg Asn Gly Thr Leu ThrVal Thr Ser Thr Leu Pro Val Gly Thr 405 410 415 Arg Asp Trp Ile Glu GlyGlu Thr Tyr Gln Cys Arg Val Thr His Pro 420 425 430 His Leu Pro Arg AlaLeu Met Arg Ser Thr Thr Ala Ser Pro Gly Lys 435 440 445 Arg Leu Ala ProGlu Val Tyr Met Leu Pro Pro Ser Pro Glu Glu Thr 450 455 460 Gly Thr ThrArg Thr Val Thr Cys Leu Ile Arg Gly Phe Tyr Pro Ser 465 470 475 480 GluIle Ser Val Gln Trp Leu Phe Asn Asn Glu Glu Asp His Thr Gly 485 490 495His His Thr Thr Thr Arg Pro Gln Lys Asp His Gly Thr Asp Pro Ser 500 505510 Phe Phe Leu Tyr Ser Arg Met Leu Val Asn Lys Ser Ile Trp Glu Lys 515520 525 Gly Asn Leu Val Thr Cys Arg Val Val His Glu Ala Leu Pro Gly Ser530 535 540 Arg Thr Leu Glu Lys Ser Leu His Tyr Ser Ala Gly Asn 545 550555 13 1041 DNA Artificial Sequence insert sequence 13 tcgagtactttatctctccc agaaagtggc cctgtgacaa tcatcccacc tacagtgaag 60 ctcttccactcatcctgtga cccccgaggg gatgctcatt ccaccatcca gctgctctgc 120 cttgtctctggcttctcccc agccaaggtc catgtgacct ggctggtaga tggacaggag 180 gctgaaaatctctttcccta tacaaccaga cctaagaggg aagggggaca gactttttct 240 ctacaaagtgaagtcaacat cacacagggc cagtggatgt catcaaacac ctacacctgc 300 catgtcaagcacaatggcag catctttgaa gacagttcta gaaagtgtgc agattccaac 360 ccgagaggggtgagcgccta cctaagccgg cccagcccgt tcgacctgtt catccgcaag 420 tcgcccacgatcacctgtct ggtggtggac ctggcaccca gcaaggggac cgtgaacctg 480 acctggtcccgggccagtgg gaagcctgtg aaccactcca ccagaaagga ggagaagcag 540 cgcaatggcacgttaaccgt cacgtccacc ctgccggtgg gcacccgaga ctggatcgag 600 ggggagacctaccagtgcag ggtgacccac ccccacctgc ccagggccct catgcggtcc 660 acgaccaagcttgctagccc aggcaaacgc ttagcccccg aggtatatat gctccctcca 720 tctccagaggaaacaggaac cactcgcact gtaacctgcc taattcgggg tttctaccct 780 tctgaaatatctgtccaatg gctgtttaat aacgaagagg accacactgg acaccatact 840 accacccgtccccaaaagga ccacggaacg gatccttcct tcttcctcta cagccgaatg 900 cttgtcaacaagtctatttg ggaaaaaggc aatctcgtca cctgccgtgt ggtgcatgaa 960 gccctacctggctcccgcac cctggaaaaa agcctgcatt actcagctgg taacggatca 1020 ggacaccatcaccatcacca t 1041 14 347 PRT Artificial Sequence chimeric polypeptide 14Ser Ser Thr Leu Ser Leu Pro Glu Ser Gly Pro Val Thr Ile Ile Pro 1 5 1015 Pro Thr Val Lys Leu Phe His Ser Ser Cys Asp Pro Arg Gly Asp Ala 20 2530 His Ser Thr Ile Gln Leu Leu Cys Leu Val Ser Gly Phe Ser Pro Ala 35 4045 Lys Val His Val Thr Trp Leu Val Asp Gly Gln Glu Ala Glu Asn Leu 50 5560 Phe Pro Tyr Thr Thr Arg Pro Lys Arg Glu Gly Gly Gln Thr Phe Ser 65 7075 80 Leu Gln Ser Glu Val Asn Ile Thr Gln Gly Gln Trp Met Ser Ser Asn 8590 95 Thr Tyr Thr Cys His Val Lys His Asn Gly Ser Ile Phe Glu Asp Ser100 105 110 Ser Arg Lys Cys Ala Asp Ser Asn Pro Arg Gly Val Ser Ala TyrLeu 115 120 125 Ser Arg Pro Ser Pro Phe Asp Leu Phe Ile Arg Lys Ser ProThr Ile 130 135 140 Thr Cys Leu Val Val Asp Leu Ala Pro Ser Lys Gly ThrVal Asn Leu 145 150 155 160 Thr Trp Ser Arg Ala Ser Gly Lys Pro Val AsnHis Ser Thr Arg Lys 165 170 175 Glu Glu Lys Gln Arg Asn Gly Thr Leu ThrVal Thr Ser Thr Leu Pro 180 185 190 Val Gly Thr Arg Asp Trp Ile Glu GlyGlu Thr Tyr Gln Cys Arg Val 195 200 205 Thr His Pro His Leu Pro Arg AlaLeu Met Arg Ser Thr Thr Lys Leu 210 215 220 Ala Ser Pro Gly Lys Arg LeuAla Pro Glu Val Tyr Met Leu Pro Pro 225 230 235 240 Ser Pro Glu Glu ThrGly Thr Thr Arg Thr Val Thr Cys Leu Ile Arg 245 250 255 Gly Phe Tyr ProSer Glu Ile Ser Val Gln Trp Leu Phe Asn Asn Glu 260 265 270 Glu Asp HisThr Gly His His Thr Thr Thr Arg Pro Gln Lys Asp His 275 280 285 Gly ThrAsp Pro Ser Phe Phe Leu Tyr Ser Arg Met Leu Val Asn Lys 290 295 300 SerIle Trp Glu Lys Gly Asn Leu Val Thr Cys Arg Val Val His Glu 305 310 315320 Ala Leu Pro Gly Ser Arg Thr Leu Glu Lys Ser Leu His Tyr Ser Ala 325330 335 Gly Asn Gly Ser Gly His His His His His His 340 345 15 1671 DNAArtificial Sequence insert sequence 15 tcgagtactt tatctctccc agaaagtggccctgtgacaa tcatcccacc tacagtgaag 60 ctcttccact catcctgtga cccccgaggggatgctcatt ccaccatcca gctgctctgc 120 cttgtctctg gcttctcccc agccaaggtccatgtgacct ggctggtaga tggacaggag 180 gctgaaaatc tctttcccta tacaaccagacctaagaggg aagggggaca gactttttct 240 ctacaaagtg aagtcaacat cacacagggccagtggatgt catcaaacac ctacacctgc 300 catgtcaagc acaatggcag catctttgaagacagttcta gaaagtgtgc agattccaac 360 ccgagagggg tgagcgccta cctaagccggcccagcccgt tcgacctgtt catccgcaag 420 tcgcccacga tcacctgtct ggtggtggacctggcaccca gcaaggggac cgtgaacctg 480 acctggtccc gggccagtgg gaagcctgtgaaccactcca ccagaaagga ggagaagcag 540 cgcaatggca cgttaaccgt cacgtccaccctgccggtgg gcacccgaga ctggatcgag 600 ggggagacct accagtgcag ggtgacccacccccacctgc ccagggccct catgcggtcc 660 acgaccaagc ttatcgatat cccagaaagtggccctgtga caatcatccc acctacagtg 720 aagctcttcc actcatcctg tgacccccgaggggatgctc attccaccat ccagctgctc 780 tgccttgtct ctggcttctc cccagccaaggtccatgtga cctggctggt agatggacag 840 gaggctgaaa atctctttcc ctatacaaccagacctaaga gggaaggggg acagactttt 900 tctctacaaa gtgaagtcaa catcacacagggccagtgga tgtcatcaaa cacctacacc 960 tgccatgtca agcacaatgg cagcatctttgaagacagtt ctagaaagtg tgcagattcc 1020 aacccgagag gggtgagcgc ctacctaagccggcccagcc cgttcgacct gttcatccgc 1080 aagtcgccca cgatcacctg tctggtggtggacctggcac ccagcaaggg gaccgtgaac 1140 ctgacctggt cccgggccag tgggaagcctgtgaaccact ccaccagaaa ggaggagaag 1200 cagcgcaatg gcacgttaac cgtcacgtccaccctgccgg tgggcacccg agactggatc 1260 gagggggaga cctaccagtg cagggtgacccacccccacc tgcccagggc cctcatgcgg 1320 tccacgaccg ctagcccagg caaacgcttagcccccgagg tatatatgct ccctccatct 1380 ccagaggaaa caggaaccac tcgcactgtaacctgcctaa ttcggggttt ctacccttct 1440 gaaatatctg tccaatggct gtttaataacgaagaggacc acactggaca ccatactacc 1500 acccgtcccc aaaaggacca cggaacggatccttccttct tcctctacag ccgaatgctt 1560 gtcaacaagt ctatttggga aaaaggcaatctcgtcacct gccgtgtggt gcatgaagcc 1620 ctacctggct cccgcaccct ggaaaaaagcctgcattact cagctggtaa c 1671 16 557 PRT Artificial Sequence chimericpolypeptide 16 Ser Ser Thr Leu Ser Leu Pro Glu Ser Gly Pro Val Thr IleIle Pro 1 5 10 15 Pro Thr Val Lys Leu Phe His Ser Ser Cys Asp Pro ArgGly Asp Ala 20 25 30 His Ser Thr Ile Gln Leu Leu Cys Leu Val Ser Gly PheSer Pro Ala 35 40 45 Lys Val His Val Thr Trp Leu Val Asp Gly Gln Glu AlaGlu Asn Leu 50 55 60 Phe Pro Tyr Thr Thr Arg Pro Lys Arg Glu Gly Gly GlnThr Phe Ser 65 70 75 80 Leu Gln Ser Glu Val Asn Ile Thr Gln Gly Gln TrpMet Ser Ser Asn 85 90 95 Thr Tyr Thr Cys His Val Lys His Asn Gly Ser IlePhe Glu Asp Ser 100 105 110 Ser Arg Lys Cys Ala Asp Ser Asn Pro Arg GlyVal Ser Ala Tyr Leu 115 120 125 Ser Arg Pro Ser Pro Phe Asp Leu Phe IleArg Lys Ser Pro Thr Ile 130 135 140 Thr Cys Leu Val Val Asp Leu Ala ProSer Lys Gly Thr Val Asn Leu 145 150 155 160 Thr Trp Ser Arg Ala Ser GlyLys Pro Val Asn His Ser Thr Arg Lys 165 170 175 Glu Glu Lys Gln Arg AsnGly Thr Leu Thr Val Thr Ser Thr Leu Pro 180 185 190 Val Gly Thr Arg AspTrp Ile Glu Gly Glu Thr Tyr Gln Cys Arg Val 195 200 205 Thr His Pro HisLeu Pro Arg Ala Leu Met Arg Ser Thr Thr Lys Leu 210 215 220 Ile Asp IlePro Glu Ser Gly Pro Val Thr Ile Ile Pro Pro Thr Val 225 230 235 240 LysLeu Phe His Ser Ser Cys Asp Pro Arg Gly Asp Ala His Ser Thr 245 250 255Ile Gln Leu Leu Cys Leu Val Ser Gly Phe Ser Pro Ala Lys Val His 260 265270 Val Thr Trp Leu Val Asp Gly Gln Glu Ala Glu Asn Leu Phe Pro Tyr 275280 285 Thr Thr Arg Pro Lys Arg Glu Gly Gly Gln Thr Phe Ser Leu Gln Ser290 295 300 Glu Val Asn Ile Thr Gln Gly Gln Trp Met Ser Ser Asn Thr TyrThr 305 310 315 320 Cys His Val Lys His Asn Gly Ser Ile Phe Glu Asp SerSer Arg Lys 325 330 335 Cys Ala Asp Ser Asn Pro Arg Gly Val Ser Ala TyrLeu Ser Arg Pro 340 345 350 Ser Pro Phe Asp Leu Phe Ile Arg Lys Ser ProThr Ile Thr Cys Leu 355 360 365 Val Val Asp Leu Ala Pro Ser Lys Gly ThrVal Asn Leu Thr Trp Ser 370 375 380 Arg Ala Ser Gly Lys Pro Val Asn HisSer Thr Arg Lys Glu Glu Lys 385 390 395 400 Gln Arg Asn Gly Thr Leu ThrVal Thr Ser Thr Leu Pro Val Gly Thr 405 410 415 Arg Asp Trp Ile Glu GlyGlu Thr Tyr Gln Cys Arg Val Thr His Pro 420 425 430 His Leu Pro Arg AlaLeu Met Arg Ser Thr Thr Ala Ser Pro Gly Lys 435 440 445 Arg Leu Ala ProGlu Val Tyr Met Leu Pro Pro Ser Pro Glu Glu Thr 450 455 460 Gly Thr ThrArg Thr Val Thr Cys Leu Ile Arg Gly Phe Tyr Pro Ser 465 470 475 480 GluIle Ser Val Gln Trp Leu Phe Asn Asn Glu Glu Asp His Thr Gly 485 490 495His His Thr Thr Thr Arg Pro Gln Lys Asp His Gly Thr Asp Pro Ser 500 505510 Phe Phe Leu Tyr Ser Arg Met Leu Val Asn Lys Ser Ile Trp Glu Lys 515520 525 Gly Asn Leu Val Thr Cys Arg Val Val His Glu Ala Leu Pro Gly Ser530 535 540 Arg Thr Leu Glu Lys Ser Leu His Tyr Ser Ala Gly Asn 545 550555 17 1698 DNA Artificial Sequence insert sequence 17 tcgagtactttatctctccc agaaagtggc cctgtgacaa tcatcccacc tacagtgaag 60 ctcttccactcatcctgtga cccccgaggg gatgctcatt ccaccatcca gctgctctgc 120 cttgtctctggcttctcccc agccaaggtc catgtgacct ggctggtaga tggacaggag 180 gctgaaaatctctttcccta tacaaccaga cctaagaggg aagggggaca gactttttct 240 ctacaaagtgaagtcaacat cacacagggc cagtggatgt catcaaacac ctacacctgc 300 catgtcaagcacaatggcag catctttgaa gacagttcta gaaagtgtgc agattccaac 360 ccgagaggggtgagcgccta cctaagccgg cccagcccgt tcgacctgtt catccgcaag 420 tcgcccacgatcacctgtct ggtggtggac ctggcaccca gcaaggggac cgtgaacctg 480 acctggtcccgggccagtgg gaagcctgtg aaccactcca ccagaaagga ggagaagcag 540 cgcaatggcacgttaaccgt cacgtccacc ctgccggtgg gcacccgaga ctggatcgag 600 ggggagacctaccagtgcag ggtgacccac ccccacctgc ccagggccct catgcggtcc 660 acgaccaagcttatcgatat cccagaaagt ggccctgtga caatcatccc acctacagtg 720 aagctcttccactcatcctg tgacccccga ggggatgctc attccaccat ccagctgctc 780 tgccttgtctctggcttctc cccagccaag gtccatgtga cctggctggt agatggacag 840 gaggctgaaaatctctttcc ctatacaacc agacctaaga gggaaggggg acagactttt 900 tctctacaaagtgaagtcaa catcacacag ggccagtgga tgtcatcaaa cacctacacc 960 tgccatgtcaagcacaatgg cagcatcttt gaagacagtt ctagaaagtg tgcagattcc 1020 aacccgagaggggtgagcgc ctacctaagc cggcccagcc cgttcgacct gttcatccgc 1080 aagtcgcccacgatcacctg tctggtggtg gacctggcac ccagcaaggg gaccgtgaac 1140 ctgacctggtcccgggccag tgggaagcct gtgaaccact ccaccagaaa ggaggagaag 1200 cagcgcaatggcacgttaac cgtcacgtcc accctgccgg tgggcacccg agactggatc 1260 gagggggagacctaccagtg cagggtgacc cacccccacc tgcccagggc cctcatgcgg 1320 tccacgaccgctagcccagg caaacgctta gcccccgagg tatatatgct ccctccatct 1380 ccagaggaaacaggaaccac tcgcactgta acctgcctaa ttcggggttt ctacccttct 1440 gaaatatctgtccaatggct gtttaataac gaagaggacc acactggaca ccatactacc 1500 acccgtccccaaaaggacca cggaacggat ccttccttct tcctctacag ccgaatgctt 1560 gtcaacaagtctatttggga aaaaggcaat ctcgtcacct gccgtgtggt gcatgaagcc 1620 ctacctggctcccgcaccct ggaaaaaagc ctgcattact cagctggtaa cggatcagga 1680 caccatcaccatcaccat 1698 18 566 PRT Artificial Sequence chimeric polypeptide 18 SerSer Thr Leu Ser Leu Pro Glu Ser Gly Pro Val Thr Ile Ile Pro 1 5 10 15Pro Thr Val Lys Leu Phe His Ser Ser Cys Asp Pro Arg Gly Asp Ala 20 25 30His Ser Thr Ile Gln Leu Leu Cys Leu Val Ser Gly Phe Ser Pro Ala 35 40 45Lys Val His Val Thr Trp Leu Val Asp Gly Gln Glu Ala Glu Asn Leu 50 55 60Phe Pro Tyr Thr Thr Arg Pro Lys Arg Glu Gly Gly Gln Thr Phe Ser 65 70 7580 Leu Gln Ser Glu Val Asn Ile Thr Gln Gly Gln Trp Met Ser Ser Asn 85 9095 Thr Tyr Thr Cys His Val Lys His Asn Gly Ser Ile Phe Glu Asp Ser 100105 110 Ser Arg Lys Cys Ala Asp Ser Asn Pro Arg Gly Val Ser Ala Tyr Leu115 120 125 Ser Arg Pro Ser Pro Phe Asp Leu Phe Ile Arg Lys Ser Pro ThrIle 130 135 140 Thr Cys Leu Val Val Asp Leu Ala Pro Ser Lys Gly Thr ValAsn Leu 145 150 155 160 Thr Trp Ser Arg Ala Ser Gly Lys Pro Val Asn HisSer Thr Arg Lys 165 170 175 Glu Glu Lys Gln Arg Asn Gly Thr Leu Thr ValThr Ser Thr Leu Pro 180 185 190 Val Gly Thr Arg Asp Trp Ile Glu Gly GluThr Tyr Gln Cys Arg Val 195 200 205 Thr His Pro His Leu Pro Arg Ala LeuMet Arg Ser Thr Thr Lys Leu 210 215 220 Ile Asp Ile Pro Glu Ser Gly ProVal Thr Ile Ile Pro Pro Thr Val 225 230 235 240 Lys Leu Phe His Ser SerCys Asp Pro Arg Gly Asp Ala His Ser Thr 245 250 255 Ile Gln Leu Leu CysLeu Val Ser Gly Phe Ser Pro Ala Lys Val His 260 265 270 Val Thr Trp LeuVal Asp Gly Gln Glu Ala Glu Asn Leu Phe Pro Tyr 275 280 285 Thr Thr ArgPro Lys Arg Glu Gly Gly Gln Thr Phe Ser Leu Gln Ser 290 295 300 Glu ValAsn Ile Thr Gln Gly Gln Trp Met Ser Ser Asn Thr Tyr Thr 305 310 315 320Cys His Val Lys His Asn Gly Ser Ile Phe Glu Asp Ser Ser Arg Lys 325 330335 Cys Ala Asp Ser Asn Pro Arg Gly Val Ser Ala Tyr Leu Ser Arg Pro 340345 350 Ser Pro Phe Asp Leu Phe Ile Arg Lys Ser Pro Thr Ile Thr Cys Leu355 360 365 Val Val Asp Leu Ala Pro Ser Lys Gly Thr Val Asn Leu Thr TrpSer 370 375 380 Arg Ala Ser Gly Lys Pro Val Asn His Ser Thr Arg Lys GluGlu Lys 385 390 395 400 Gln Arg Asn Gly Thr Leu Thr Val Thr Ser Thr LeuPro Val Gly Thr 405 410 415 Arg Asp Trp Ile Glu Gly Glu Thr Tyr Gln CysArg Val Thr His Pro 420 425 430 His Leu Pro Arg Ala Leu Met Arg Ser ThrThr Ala Ser Pro Gly Lys 435 440 445 Arg Leu Ala Pro Glu Val Tyr Met LeuPro Pro Ser Pro Glu Glu Thr 450 455 460 Gly Thr Thr Arg Thr Val Thr CysLeu Ile Arg Gly Phe Tyr Pro Ser 465 470 475 480 Glu Ile Ser Val Gln TrpLeu Phe Asn Asn Glu Glu Asp His Thr Gly 485 490 495 His His Thr Thr ThrArg Pro Gln Lys Asp His Gly Thr Asp Pro Ser 500 505 510 Phe Phe Leu TyrSer Arg Met Leu Val Asn Lys Ser Ile Trp Glu Lys 515 520 525 Gly Asn LeuVal Thr Cys Arg Val Val His Glu Ala Leu Pro Gly Ser 530 535 540 Arg ThrLeu Glu Lys Ser Leu His Tyr Ser Ala Gly Asn Gly Ser Gly 545 550 555 560His His His His His His 565 19 1035 DNA Artificial Sequence insertsequence 19 gaattcgtca acaaaacctt tggcgtcagc tccaggaact tcaccccacctaccgtgaag 60 atcttacagt catcctgcaa tgacgacggg cactttcccc cgaccatccagctcctgtgc 120 ctcatctccg ggtacacccc aggggccatc aatgtcacct ggctggagaacgggcaggtc 180 atgaaagtga actcgcccac ccctcctgcc acgcaggagg gtgagctggcctccacacaa 240 agtgagttca ccctcgccca gaagcactgg ctgtcggacc gcacttacacctgccaggtc 300 acctatcaag gtaccaccta taacgacagc accaagaagt gtgcagattccaacccgaga 360 ggggtgagtg cctacctaag ccggcccagc ccgtttgacc tgttcatcagcaagtcgccc 420 acgatcacct gtctggtggt ggacctggca cccagcaagg agaccgtgaacctgacctgg 480 tcccgggcca gtgggaagcc tgtgccccac atccccgcaa cggggaagaagcagcgcaat 540 ggcacgttaa ccgttacgtc catcctgccg gtggtcaccc aagactggatcgagggggag 600 acctaccagt gcagggtgac ccacccccac ctccccaggg ccctcgtgcggtccatgacc 660 aagaccagcg gcccgcgtgc tgccccggaa gtctatgtgt ttgcaacgccagagaagcta 720 gagagccggg acaagcgcac cctcgcctgc ctgatccaga acttcatgcctgaggacata 780 tcggtgcagt ggctgcacag cgacgtgcag ctcccggacg cccggcacagcgtgacgcag 840 ccccgcaaga ccaagggctc cggcttcttc gtcttcagcc gcctggaggtgaccaaggcc 900 gaatgggagc agaaagacga gttcatctgc cgtgcagtcc atgaggcagcgagcccctca 960 tggatcgtcc agcaagcggt gtctgtaaat cccggtaaag gatcaggacaccatcaccat 1020 caccattgac tcgag 1035 20 1077 DNA Artificial Sequenceinsert sequence 20 gaattccacc atcaccatca ccatacttta tctctcccagaaagtggccc tgtgacaatc 60 atcccaccta cagtgaagct cttccactca tcctgtgacccccgagggga tgctcattcc 120 accatccagc tgctctgcct tgtctctggc ttctccccagccaaggtcca tgtgacctgg 180 ctggtagatg gacaggaggc tgaaaatctc tttccctatacaaccagacc taagagggaa 240 gggggacaga ctttttctct acaaagtgaa gtcaacatcacacagggcca gtggatgtca 300 tcaaacacct acacctgcca tgtcaagcac aatggcagcatctttgaaga cagttctaga 360 aagtgtgcag attccaaccc gagaggggtg agtgcctacctaagccggcc cagcccgttt 420 gacctgttca tcagcaagtc gcccacgatt acctgtctggtggtggacct ggcacccagc 480 aaggagaccg tgaacctgac ctggtcccgg gccagtgggaagcctgtgcc ccacatcccc 540 gcaacgggga agaagcagcg caatggcacg ttaaccgttacgtccatcct gccggtggtc 600 acccaagact ggatcgaggg ggagacctac cagtgcagggtgacccaccc ccacctcccc 660 agggccctcg tgcggtccat gaccaagctt gctagcccaggcaaacgctt agcccccgag 720 gtatatatgc tccctccatc tccagaggaa acaggaaccactcgcactgt aacctgccta 780 attcggggtt tctacccttc tgaaatatct gtccaatggctgtttaataa cgaagaggac 840 cacactggac accatactac cacccgtccc caaaaggaccacggaacgga tccttccttc 900 ttcctctaca gccgaatgct tgtcaacaag tctatttgggaaaaaggcaa tctcgtcacc 960 tgccgtgtgg tgcatgaagc cctacctggc tcccgcaccctggaaaaaag cctgcattac 1020 tcagctggta acggatcagg acaccatcac catcaccattgattaccctg actcgag 1077 21 353 PRT Artificial Sequence chimericpolypeptide 21 Glu Phe His His His His His His Thr Leu Ser Leu Pro GluSer Gly 1 5 10 15 Pro Val Thr Ile Ile Pro Pro Thr Val Lys Leu Phe HisSer Ser Cys 20 25 30 Asp Pro Arg Gly Asp Ala His Ser Thr Ile Gln Leu LeuCys Leu Val 35 40 45 Ser Gly Phe Ser Pro Ala Lys Val His Val Thr Trp LeuVal Asp Gly 50 55 60 Gln Glu Ala Glu Asn Leu Phe Pro Tyr Thr Thr Arg ProLys Arg Glu 65 70 75 80 Gly Gly Gln Thr Phe Ser Leu Gln Ser Glu Val AsnIle Thr Gln Gly 85 90 95 Gln Trp Met Ser Ser Asn Thr Tyr Thr Cys His ValLys His Asn Gly 100 105 110 Ser Ile Phe Glu Asp Ser Ser Arg Lys Cys AlaAsp Ser Asn Pro Arg 115 120 125 Gly Val Ser Ala Tyr Leu Ser Arg Pro SerPro Phe Asp Leu Phe Ile 130 135 140 Ser Lys Ser Pro Thr Ile Thr Cys LeuVal Val Asp Leu Ala Pro Ser 145 150 155 160 Lys Glu Thr Val Asn Leu ThrTrp Ser Arg Ala Ser Gly Lys Pro Val 165 170 175 Pro His Ile Pro Ala ThrGly Lys Lys Gln Arg Asn Gly Thr Leu Thr 180 185 190 Val Thr Ser Ile LeuPro Val Val Thr Gln Asp Trp Ile Glu Gly Glu 195 200 205 Thr Tyr Gln CysArg Val Thr His Pro His Leu Pro Arg Ala Leu Val 210 215 220 Arg Ser MetThr Lys Leu Ala Ser Pro Gly Lys Arg Leu Ala Pro Glu 225 230 235 240 ValTyr Met Leu Pro Pro Ser Pro Glu Glu Thr Gly Thr Thr Arg Thr 245 250 255Val Thr Cys Leu Ile Arg Gly Phe Tyr Pro Ser Glu Ile Ser Val Gln 260 265270 Trp Leu Phe Asn Asn Glu Glu Asp His Thr Gly His His Thr Thr Thr 275280 285 Arg Pro Gln Lys Asp His Gly Thr Asp Pro Ser Phe Phe Leu Tyr Ser290 295 300 Arg Met Leu Val Asn Lys Ser Ile Trp Glu Lys Gly Asn Leu ValThr 305 310 315 320 Cys Arg Val Val His Glu Ala Leu Pro Gly Ser Arg ThrLeu Glu Lys 325 330 335 Ser Leu His Tyr Ser Ala Gly Asn Gly Ser Gly HisHis His His His 340 345 350 His 22 1734 DNA Artificial Sequence insertsequence 22 gaattccacc atcaccatca ccatacttta tctctcccag aaagtggccctgtgacaatc 60 atcccaccta cagtgaagct cttccactca tcctgtgacc cccgaggggatgctcattcc 120 accatccagc tgctctgcct tgtctctggc ttctccccag ccaaggtccatgtgacctgg 180 ctggtagatg gacaggaggc tgaaaatctc tttccctata caaccagacctaagagggaa 240 gggggacaga ctttttctct acaaagtgaa gtcaacatca cacagggccagtggatgtca 300 tcaaacacct acacctgcca tgtcaagcac aatggcagca tctttgaagacagttctaga 360 aagtgtgcag attccaaccc gagaggggtg agtgcctacc taagccggcccagcccgttt 420 gacctgttca tcagcaagtc gcccacgatt acctgtctgg tggtggacctggcacccagc 480 aaggagaccg tgaacctgac ctggtcccgg gccagtggga agcctgtgccccacatcccc 540 gcaacgggga agaagcagcg caatggcacg ttaaccgtta cgtccatcctgccggtggtc 600 acccaagact ggattgaggg ggagacctac cagtgcaggg tgacccacccccacctcccc 660 agggccctcg tgcggtccat gaccaagctt atcgatatcc cagaaagtggccctgtgaca 720 atcatcccac ctacagtgaa gctcttccac tcatcctgtg acccccgaggggatgctcat 780 tccaccatcc agctgctctg ccttgtctct ggcttctccc cagccaaggtccatgtgacc 840 tggctggtag atggacagga ggctgaaaat ctctttccct atacaaccagacctaagagg 900 gaagggggac agactttttc tctacaaagt gaagtcaaca tcacacagggccagtggatg 960 tcatcaaaca cctacacctg ccatgtcaag cacaatggca gcatctttgaagacagttct 1020 agaaagtgtg cagattccaa cccgagaggg gtgagtgcct acctaagccggcccagcccg 1080 tttgacctgt tcatcagcaa gtcgcccacg attacctgtc tggtggtggacctggcaccc 1140 agcaaggaga ccgtgaacct gacctggtcc cgggccagtg ggaagcctgtgccccacatc 1200 cccgcaacgg ggaagaagca gcgcaatggc acgttaaccg ttacgtccatcctgccggtg 1260 gtcacccaag actggattga gggggagacc taccagtgca gggtgacccacccccacctc 1320 cccagggccc tcgtgcggtc catgaccgct agcccaggca aacgcttagcccccgaggta 1380 tatatgctcc ctccatctcc agaggaaaca ggaaccactc gcactgtaacctgcctaatt 1440 cggggtttct acccttctga aatatctgtc caatggctgt ttaataacgaagaggaccac 1500 actggacacc atactaccac ccgtccccaa aaggaccacg gaacggatccttccttcttc 1560 ctctacagcc gaatgcttgt caacaagtct atttgggaaa aaggcaatctcgtcacctgc 1620 cgtgtggtgc atgaagccct acctggctcc cgcaccctgg aaaaaagcctgcattactca 1680 gctggtaacg gatcaggaca ccatcaccat caccattgat taccctgactcgag 1734 23 20 DNA Artificial Sequence target sequence 23 aggtcgtgtactgtcagtca 20 24 20 DNA Artificial Sequence identified sequence 24acgtggtgaa ctgccagtga 20

What is claimed is:
 1. A composition comprising a polypeptide and analuminum compound, wherein said polypeptide comprises a self IgEpolypeptide sequence, and wherein administration of said composition toa mammal reduces the level of detectable free IgE in said mammal.
 2. Thecomposition of claim 1, wherein said polypeptide is a chimeric IgEpolypeptide.
 3. The composition of claim 1, wherein said polypeptidecomprises a sequence set forth in SEQ ID NO:3, SEQ ID NO:6, SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, or SEQ IDNO:21.
 4. The composition of claim 1, wherein said composition comprisesbetween about ten micrograms and about one gram of said polypeptide. 5.The composition of claim 1, wherein said composition comprises about 280micrograms of said polypeptide.
 6. The composition of claim 1, whereinsaid aluminum compound is an aluminum hydrogel compound.
 7. Thecomposition of claim 1, wherein said aluminum compound is alum.
 8. Thecomposition of claim 7, wherein said composition comprises between aboutten microliters and about one milliliter of said alum.
 9. Thecomposition of claim 7, wherein said composition comprises about 50microliters of said alum.
 10. The composition of claim 1, wherein saidreduction is at least about a 10 percent reduction.
 11. The compositionof claim 1, wherein said reduction is at least about a 30 percentreduction.
 12. The composition of claim 1, wherein said reduction is areduction from about 10 percent to about 95 percent.
 13. The compositionof claim 1, wherein said reduction is a reduction from about 20 percentto about 95 percent.
 14. The composition of claim 1, wherein saidreduction is detectable in an ELISA.
 15. The composition of claim 14,wherein an IgE receptor polypeptide sequence is used in said ELISA. 16.The composition of claim 1, wherein said administration of saidcomposition to said mammal produces an anti self IgE antibody responsewith a titer dilution₅₀ value greater than
 100. 17. The composition ofclaim 16, wherein said titer dilution₅₀ value is greater than
 200. 18.The composition of claim 16, wherein said titer dilution₅₀ value isgreater than
 400. 19. A composition comprising a polypeptide and MN51,wherein said polypeptide contains a self IgE polypeptide sequence, andwherein administration of said composition to a mammal reduces the levelof detectable free IgE in said mammal.
 20. The composition of claim 19,wherein said polypeptide is a chimeric IgE polypeptide.
 21. Thecomposition of claim 19, wherein said polypeptide comprises a sequenceset forth in SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:12, SEQID NO:14, SEQ ID NO:16, SEQ ID NO:18, or SEQ ID NO:21.
 22. Thecomposition of claim 19, wherein said composition comprises betweenabout ten micrograms and about one gram of said polypeptide.
 23. Thecomposition of claim 19, wherein said composition comprises about 100micrograms of said polypeptide.
 24. The composition of claim 19, whereinsaid composition comprises between about ten microliters and about onemilliliter of said MN51.
 25. The composition of claim 19, wherein saidcomposition comprises about 50 microliters of said MN51.
 26. Thecomposition of claim 19, wherein said reduction is at least about a 10percent reduction.
 27. The composition of claim 19, wherein saidreduction is at least about a 30 percent reduction.
 28. The compositionof claim 19, wherein said reduction is a reduction from about 10 percentto about 95 percent.
 29. The composition of claim 19, wherein saidreduction is a reduction from about 20 percent to about 95 percent. 30.The composition of claim 19, wherein said reduction is detectable in anELISA.
 31. The composition of claim 30, wherein an IgE receptorpolypeptide sequence is used in said ELISA.
 32. The composition of claim19, wherein said administration of said composition to said mammalproduces an anti self IgE antibody response with a titer dilution₅₀value greater than
 100. 33. The composition of claim 32, wherein saidtiter dilution₅₀ value is greater than
 200. 34. The composition of claim32, wherein said titer dilution₅₀ value is greater than
 400. 35. Acomposition comprising an aluminum compound and about 30 to 300micrograms of a chimeric IgE polypeptide.
 36. A composition comprisingMN51 and about 30 to 300 micrograms of a chimeric IgE polypeptide.
 37. Amethod for inducing an anti self IgE antibody response in a mammal, saidmethod comprising administering to said mammal a composition underconditions wherein said mammal reduces the level of detectable free IgEin said mammal, wherein said composition comprises a polypeptide and analuminum compound, and wherein said polypeptide comprises a selfpolypeptide sequence.
 38. The method of claim 37, wherein saidpolypeptide comprises an amino acid sequence set forth in SEQ ID NO:3,SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQID NO:18, or SEQ ID NO:21.
 39. A method for inducing an anti self IgEantibody response in a mammal, said method comprising administering tosaid mammal a composition under conditions wherein said mammal reducesthe level of detectable free IgE in said mammal, wherein saidcomposition comprises a polypeptide and MN51, and wherein saidpolypeptide contains a self polypeptide sequence.
 40. The method ofclaim 39, wherein said polypeptide comprises an amino acid sequence setforth in SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:12, SEQ IDNO:14, SEQ ID NO:16, SEQ ID NO:18, or SEQ ID NO:21.
 41. A method forinducing a reversible anti self-IgE response in a primate, said methodcomprising administering a polypeptide having a self IgE sequence tosaid primate under conditions wherein said primate mounts an antibodyresponse to self-IgE that peaks and then decreases with time.
 42. Themethod of claim 41, wherein said primate is a monkey.
 43. The method ofclaim 41, wherein said antibody response to self-IgE is a primaryresponse that decreases with time.
 44. The method of claim 41, whereinsaid antibody response to self-IgE decreases to undetectable levelswithin nine months of said administration.
 45. The method of claim 41,wherein said polypeptide comprises a sequence set forth in SEQ ID NO:6,SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, orSEQ ID NO:21.
 46. A method for inducing an anti self-IgE response in amammal after said mammal has experienced a primary anti self-IgEresponse, said method comprising administering a polypeptide having aself IgE sequence to said mammal under conditions wherein said mammalmounts an antibody response to self-IgE in a manner consistent with asecondary antibody response.
 47. The method of claim 46, wherein saidmammal is a primate.
 48. The method of claim 46, wherein saidpolypeptide comprises a sequence set forth in SEQ ID NO:3, SEQ ID NO:6,SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, orSEQ ID NO:21.
 49. A method for inducing a series of anti self-IgEresponses in a mammal, said method comprising administering apolypeptide having a self IgE sequence to said mammal at different timesand under conditions wherein said mammal mounts a detectable antiself-IgE response that peaks within at least one year of eachadministration.
 50. The method of claim 49, wherein said mammal mounts adetectable anti self-IgE response that peaks within at least threemonths of each administration.